Neurodegenerative disorders

ABSTRACT

A cyclic polypeptide, derivative or analogue thereof, comprising an amino acid sequence derived from the C-terminus of acetylcholinesterase (ACh E), or a truncation thereof.

The invention relates to neurodegenerative disorders, and in particularto novel compositions, therapies and methods for treating suchconditions, for example Alzheimer's disease.

Alzheimer's disease primarily affects men and women over the age of 65and the likelihood of being diagnosed with the disease increasessubstantially with age. With the percentage of adults over the age of 65expected to grow worldwide over the next 40 years, the incidence ofAlzheimer's disease is expected to more than double, escalating from 21million cases in 2010 to 53 million in 2050 (statistics fromwww.alzheimersresearchuk.org and www.alz.org). This exponential increasein the expected number of patients presenting with Alzheimer's diseasenot only represents a major area of unmet medical need, but offers asignificant market opportunity for therapeutics and diagnostics as thereis currently no fully effective method of treating the disease.

There has been no new drug to combat Alzheimer's disease specifically,nor neurodegeneration more generally, in the last 10 years. The reasonis that as yet, the basic underlying brain mechanism has not yet beenidentified that could consequently be targeted pharmaceutically. Themain contender for accounting for the process of neurodegeneration isthe ‘amyloid hypothesis’, where neuronal death is attributed todisruption of the cell membrane by toxic deposits of amyloid,characteristic of post-mortem Alzheimer brain, and resulting fromabnormal cleavage of amyloid precursor protein. However, this ‘amyloidhypothesis’ does not explain the co-pathology frequently observed withAlzheimer's and Parkinson's diseases, nor the characteristic selectivityof cells vulnerable to degeneration, nor the absence of amyloid depositsin animal models of dementia, nor indeed the occurrence of amyloid incertain brain regions where cognitive deficits are not apparent. Despitethe popularity of amyloid formation as a pharmaceutical target over thelast two decades, no treatment based on this theory has as yet provedeffective. A more likely possibility is that once the neurodegenerativeprocess is underway, then amyloid will additionally be generated as asecondary, exacerbating effect that is less specific.

One clue for identifying the primary mechanism of neurodegeneration,could be that only various neuronal groups are primarily vulnerable.Moreover, the diverse cell sub-groups prone to Alzheimer's, Parkinson'sand Motor Neurone Diseases nonetheless are adjacent to each other andform a continuous ‘hub’ extending from brainstem to forebrain that allsend diffuse projections upwards and outwards to higher cerebralcentres. Hence, despite their heterogeneity in transmitters, theseneuronal groups have been collectively dubbed ‘Global’ neurons todistinguish them from the more familiar and localised circuits of cellsin most other parts of the brain, such as cerebellum, thalamus, cortexetc. These selectively vulnerable Global neurons were previouslyidentified, albeit using a different terminology (‘isodendritic core’)as pivotal in neurodegeneration several decades ago.

The sub-groups of Global neurons have a specific feature in common thatmight explain the puzzling and as yet unanswered question as to why onlythese cells succumb to progressive death whilst their counterpartselsewhere in the brain, even when damaged by stroke, do not: they retaina robust plasticity into and throughout adulthood, accompanied by aspecific sensitivity to substances aiding and sustaining growth-‘trophicfactors’. In the developing brain, trophic factors work by stimulatingcalcium influx, which triggers a cascade of events within the cell,eventually resulting in selective differentiation and growth. However,in higher doses or with longer exposures, sustained calcium entry can betoxic to neurons. Most significantly, a further determining factor inwhether or not calcium entry triggers trophic or toxic effects, is age:as neurons mature, an erstwhile trophic level of intracellular calciumbecomes lethal.

The inventor has previously proposed that the neurodegenerative processis in fact an aberrantly activated process of development. In support ofthis hypothesis, a hyper-trophy of the brainstem ‘hub’ neurons hasactually been reported in Alzheimer brains (Bowser et al., 1997, BrainPathol. 7:723-30). If large areas of this hub are damaged, then morethan one neurodegenerative disease will present, as occurs in thefrequently seen but never as yet explained cases of co-pathology withAlzheimer's and Parkinson's diseases. Interestingly, all the neuronswithin the vulnerable hub of Global neurons, despite transmitterheterogeneity, all contain the familiar enzyme acetylcholinesterase(AChE). AChE is therefore present in neurons where it would be unable toperform its normal function, since such sub-groups of cells as thenoradrenergic locus coeruleus, the dopaminergic substantia nigra, or theserotonergic raphe nuclei, in no cases contain the usual substrate,acetylcholine. A further unexpected deviation from its normal, enzymaticrole is that the AChE is actually released from Global neurons,presumably as some kind of inter-cellular messenger in its own right. Ingeneral, AChE is now widely and well-established as a signallingmolecule that has trophic activity in a diverse variety of situations inboth neural and non-neural tissue.

The inventor has previously shown that AChE, operating as a trophicagent independent of its enzymatic action, does indeed trigger calciumentry into neurons. It is possible therefore that within Global neurons,AChE has a dual non-classical action that ranges along a trophic-toxicaxis, depending on amount, duration of availability and, mostsignificantly, age. If standard neurons are damaged in adulthood, as ina stroke, others will compensate functionally. In contrast, Globalneurons will respond by calling on their trophic resources in an attemptto regenerate. But because the subsequent calcium influx will be lethalin the older, mature cells, the resulting damage will trigger furtherattempts to compensate in a pernicious cycle that characterisesneurodegeneration.

Acetylcholinesterase (AChE) is expressed at different stages ofdevelopment in various forms, all of which have identical enzymaticactivity, but which have very different molecular composition. The‘tailed’ (T-AChE) is expressed at synapses and the inventors havepreviously identified two peptides that could be cleaved from theC-terminus, one referred to as “T14”, within the other which is known as“T30”, and which both have strong sequence homology to the comparableregion of β-amyloid (see FIG. 11; and SEQ ID NO's: 2-5). The AChEC-terminal peptide “T14” has been identified as being the salient partof the AChE molecule responsible for its range of non-hydrolyticactions. The synthetic 14 amino acids peptide analogue (i.e. “T14”), andsubsequently the larger, more stable, and more potent amino acidsequence in which it is embedded (i.e. “T30”) display actions comparableto those reported for ‘non-cholinergic’ AChE, where the inert residuewithin the T30 sequence (i.e. “T15”) is without effect.

Acute effects of T14 and T30 are that they:—(i) modulate calcium entryinto neurons in brain slices over time scales from milliseconds tohours; (ii) compromise cell viability in PC 12 cells and also inneuronal organotypic cultures in vitro. (iii) modulate ‘compensatory’calcium-induced AChE release from neurons and PC 12 cells; (iv) activatecalcium currents in oocytes and neurons in brain slices; (v) synergisewith amyloid in toxic effects; and (vi) are involved in amyloidprecursor protein production and amyloid beta peptide release. Chroniceffects of T14 and T30 are that they:—(i) reduce neuron growth; (ii)induce apoptosis; (iii) increase AChE release; (iv) bind to and modulateα7 nicotinic-receptor; and (v) enhance expression of the α7 receptor onthe cell surface over 24 hours, thereby providing a feedforwardmechanism for further toxicity.

Since T14 and T30 are more selective than β-amyloid in inducing toxicityand are also synergistic with amyloid exacerbating toxicity, it has beenpostulated that any agent blocking the effect of T14 or T30 would alsoreduce the less selective and subsequent toxic effect of amyloid. Theinventor has previously shown that T30 and T14 peptides bind to anallosteric site on the α7 nicotinic-receptor to induce a spectrum oftrophic-toxic effects. This receptor is co-expressed with AChE duringcritical periods of brain development as well as showing a closelyparallel distribution in the adult brain, and is one of the mostpowerful calcium ionophores in the brain. It can also functionindependent of cholinergic transmission, since choline (derived fromdiet) can serve as an alternative primary ligand. Moreover, thisreceptor has already been implicated in Alzheimer's disease as one ofthe targets for the current therapy galanthamine (Reminyl®), as well asbeing linked to the actions of amyloid.

However, the efficacy of galanthamine has proved limited, whilst otherα7 nicotinic acetylcholine receptor antagonists are still in clinicaltrials. Galanthamine has a low affinity for the α7 nicotinic-receptor(i.e. only 10 μM) compared to that of T30 and T14, which have muchhigher affinities for the α7 nicotinic-receptor (i.e. 5 nM). Hence if,in an Alzheimer's brain, the endogenous equivalent of T30 peptide isalready occupying the respective receptor site, galanthamine would needto be given at non-physiological, high doses with inevitable sideeffects and most importantly, questionable efficacy.

There is therefore a need to provide an improved medicament for thetreatment of neurodegenerative disorders, such as Alzheimer's diseaseand Parkinson's disease.

As described in the Examples, the inventor has surprisingly demonstratedthat cyclic forms of peptides derived from the C-terminus of AChE can beused to selectively inhibit the non-classical effects of AChE and/or itsterminal peptide in vitro (i.e. the effects of AChE that are independentof its enzymatic activity), and therefore effectively treatneurodegenerative disorders.

Thus, according to a first aspect of the invention, there is provided acyclic polypeptide, derivative or analogue thereof, comprising an aminoacid sequence derived from the C-terminus of acetylcholinesterase(AChE), or a truncation thereof.

Cyclic polypeptides are peptide chains whose N- and C-termini arethemselves linked together with a peptide bond that forms a circularchain of amino acids, and, to date, no cyclic peptides have beendeveloped which comprise an amino acid sequence derived from theC-terminus of acetylcholinesterase (AChE), or a truncation thereof. Asdescribed in the Examples, the inventor has surprisingly demonstratedthat the inefficacy of protection against the non-specific action ofhydrogen peroxide would suggest that the blocking action of the cyclicpolypeptide of the first aspect is highly selective, andreceptor-mediated. The inventor was also very surprised to observe thatthe cyclic polypeptides of the invention antagonise the toxic effects ofthe known linear peptides, T14 and T30, in a variety of tests indicatingthat they prevent the additional influx of calcium through an allostericsite (e.g. an Ivermectin-sensitive allosteric site) of the α7nicotinic-receptor and effectively outcompete binding for the linear T14and T30 peptides, as well as β-amyloid. Therefore, the cyclicpolypeptide, derivative or analogue thereof may be a selectiveantagonist of the α7 nicotinic-receptor.

However, the inventors have shown that the cyclic polypeptides of theinvention act as an inert allosteric modulator of the α7nicotinic-receptor which antagonises the action of T30 and amyloid betapeptides. Therefore, preferably the cyclic polypeptide, derivative oranalogue thereof is a selective allosteric modulator of the α7nicotinic-receptor, more preferably an inert selective allostericmodulator thereof. The term “inert” can mean that the polypeptide of theinvention only acts as an allosteric modulator of the receptor in thepresence of the toxic compounds, i.e. T30 and amyloid beta peptides(β-amyloid).

Preferably, the cyclic polypeptide, derivative or analogue thereofprevents the additional influx of calcium through an allosteric site(most preferably, an Ivermectin-sensitive allosteric site) of the α7nicotinic-receptor. It is preferred that the cyclic polypeptide,derivative or analogue thereof outcompetes binding for β-amyloid.

It could not have been predicted that the peptides of the inventionwould outcompete the endogenous equivalent of T30 peptide alreadyoccupying the respective receptor site. Furthermore, the enhancedstability of cyclic peptides would account for this effectivedisplacement. Accordingly, the cyclic polypeptide prevents thepreviously established toxic effects of the linear T14, T30 peptides andalso β-amyloid. The inventor believes therefore that the cyclicpolypeptides of the invention will have significant utility for thetreatment of neurodegenerative disorders in stabilising any further cellloss.

The term “derivative or analogue thereof” can mean a polypeptide withinwhich amino acid residues are replaced by residues (whether naturalamino acids, non-natural amino acids or amino acid mimics) with similarside chains or peptide backbone properties.

The term “derived from” can mean an amino acid sequence which is aderivative or a modification of an amino acid sequence that is presentin, or forms, the C-terminus of AChE, and portion thereof.

The term “truncation thereof” can mean the cyclic polypeptide derivedfrom AChE is reduced in size by the removal of amino acids. Thereduction of amino acids may be achieved by removal of residues from theC- or N-terminal of the peptide prior to cyclisation into the cyclicpolypeptide of the invention, or may be achieved by deletion of one ormore amino acids from within the core of the peptide prior tocyclisation.

Preferably, the cyclic polypeptide is purified and/or isolated, i.e. itis not found in nature.

Acetylcholinesterase is a serine protease that hydrolyses acetylcholine,and will be well-known to the skilled person. The major form ofacetylcholinesterase which is found in the brain is known as tailedacetylcholinesterase (T-AChE). Given that the invention is primarilyconcerned with treating neurodegenerative disorders, it is preferredthat the cyclic polypeptide, derivative or analogue thereof comprises anamino acid sequence derived from the C-terminus of tailedacetylcholinesterase (T-AChE), or a truncation thereof.

The protein sequence of one embodiment of human tailedacetylcholinesterase (Gen Bank: AAA68151.1) is 614 amino acids inlength, and is provided herein as SEQ ID No:1, as follows:

[SEQ ID No: 1]   1mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv  61saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn 121relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm 181nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv 241gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel 301vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg 361vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe 421dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy 481eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq 541yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn 601qfdhyskqdr csdl

It will be appreciated that the first 31 amino acid residues of SEQ IDNo:1 are removed while the protein is released, thereby leaving a 583amino acid sequence. Accordingly, it is preferred that the cyclicpolypeptide, derivative or analogue thereof comprises an amino acidsequence derived from the C-terminus of acetylcholinesterase, or atruncation thereof, wherein the acetylcholinesterase comprises an aminoacid sequence substantially as set out in SEQ ID No:1, preferablyexcluding the 31 amino acids at the N-terminal.

Preferably, the cyclic polypeptide, derivative or analogue thereofcomprises an amino acid sequence derived from the last 300, 200, 100 or50 amino acids forming the C-terminus of acetylcholinesterase, or atruncation thereof, preferably wherein the acetylcholinesterasecomprises an amino acid sequence substantially as set out in SEQ IDNo:1. The cyclic polypeptide, derivative or analogue thereof preferablycomprises an amino acid sequence derived from the last 40 amino acidsforming the C-terminus of acetylcholinesterase, or a truncation thereof.

Preferably, the cyclic polypeptide, derivative or analogue thereofcomprises between 8 and 40 amino acid residues, more preferably between10 and 30 amino acids, and most preferably between 12 and 20 aminoacids. As shown in Figure ii, the inventor has prepared a sequencealignment between β-amyloid (Aβ), and three peptides that are derivedfrom the C-terminus of AChE, which are referred to herein as T30, T14and T15 The amino acid sequence of part of β-amyloid (Aβ) is providedherein as SEQ ID No:2, as follows:—

[SEQ ID No: 2] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

The amino acid sequence of T30 (which corresponds to the last 30 aminoacid residues of SEQ ID No:1) is provided herein as SEQ ID No:3, asfollows:—

[SEQ ID No: 3] KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

The amino acid sequence of T14 (which corresponds to the 14 amino acidresidues located towards the end of SEQ ID No:1, and lacks the final 15amino acids found in T30) is provided herein as SEQ ID No:4, asfollows:—

[SEQ ID No: 4] AEFHRWSSYWVHWK

The amino acid sequence of T15 (which corresponds to the last 15 aminoacid residues of SEQ ID No:1) is provided herein as SEQ ID No:5, asfollows:—

[SEQ ID No: 5] NQFDHYSKQDRCSDL

The inventor has generated a consensus sequence based on SEQ ID No's2-5, which is provided herein as SEQ ID No:6, as follows:—

[SEQ ID No: 6] AEFx₁x₂x₃Sx₄Yx₅vH

Preferably, in SEQ ID No:6, x₁ can be a basic amino acid residue,preferably histidine (H); x₂ can be a basic amino acid residue,preferably arginine (R); x₃ can be an aromatic amino acid residue,preferably tryptophan (W); x₄ can be an amino acid residue having analiphatic hydroxyl side chain, preferably serine (S); x₅ can betryptophan (W) or methionine (M).

It will be appreciated that any of the sequences represented as SEQ IDNo:2-6 can be readily cyclated to form a cyclic polypeptide of the firstaspect. For example, cyclization of peptides can be achieved bysidechain-to-sidechain, sidechain-to-backbone, or head-to-tail(C-terminus to N-terminus) cyclization techniques. In one preferredembodiment, head-to-tail cyclization is the preferred method by whichthe cyclic polypeptides are produced. The cyclic polypeptides may besynthesised using either classical solution-phase linear peptidecyclization or resin-based cyclization. Preferred methods forcyclization are described in the Examples. In another preferredembodiment, the polypeptide is produced using a cyclization cleavageapproach, in which the cyclic polypeptide is synthesized by cyclizationafter step-wise linear peptide synthesis. An advantage of this method isthat the sidechain does not need to be anchored, making the approachmore general. Preferably, prior to use, resultant samples of cyclicpeptides can be analysed by MALDI-TOF MS.

Accordingly, a preferred polypeptide according to the inventioncomprises cyclic SEQ ID No:3, 4, 5 or 6, or a functional variant orfragment thereof.

In one embodiment, the cyclic polypeptide comprises the amino acidsequence of SEQ ID No:3, and the N-terminal lysine residue is linked tothe C-terminal leucine residue to form a circular chain of amino acids.In another embodiment, the cyclic polypeptide comprises the amino acidsequence of SEQ ID No:4, and the N-terminal alanine residue is linked tothe C-terminal lysine residue. In yet another embodiment, the cyclicpolypeptide comprises the amino acid sequence of SEQ ID No:5, and theN-terminal asparagine residue is linked to the C-terminal leucineresidue. In another embodiment, the cyclic polypeptide comprises theamino acid sequence of SEQ ID No:6, wherein x₁ can be a basic amino acidresidue, preferably histidine (H); x₂ can be a basic amino acid residue,preferably arginine (R); x₃ can be an aromatic amino acid residue,preferably tryptophan (W); x₄ can be an amino acid residue having analiphatic hydroxyl side chain, preferably serine (S); x₅ can betryptophan (W) or methionine (M), and the N-terminal alanine residue islinked to the C-terminal histidine residue.

The inventor found that cyclated SEQ ID No: 4 (i.e. referred to hereinas “cyclated T14”, “CT14” or “NBP14”) surprisingly acts as a trueantagonist of the α7 nicotinic-receptor, i.e. that cyclated SEQ ID No:4protects cells from linear T14, T30 and β-amyloid toxicity. Moreover,cyclated T14 blocks compensatory AChE release induced by this toxicityof linear T14 and T30. In addition, they observed that cyclic T14 givenalone has no significant effects on Ca²⁺ concentrations in rat brainslices, but blocks the effects of β-amyloid. Accordingly, a preferredcyclic polypeptide of the first aspect comprises cyclic SEQ ID No:4, ora functional variant or fragment thereof.

The skilled person would appreciate that functional variants andanalogues retain substantially the same biological activity as cyclicT14 in any of the experiments described in the Examples. Accordingly, afunctional variant or analogue may be selected on the basis of itsantagonistic activity at the Ivermectin-sensitive allosteric site on theα7 nicotinic-receptor, or by the extent to which it blocks AChE release,or the extent to which it protects cells from linear T14, T30 andβ-amyloid toxicity, or the extent to which it modulates Ca²⁺ levels in arat brain slice.

The inventor is of the firm view that observed receptor antagonismprovided by cyclation of the polypeptide, derivative or analogue thereofaccording to the first aspect was so surprising that it could never havebeen obvious to the skilled person. As such, the inventor believes thatcyclation of any polypeptide could be used for antagonising a receptor,such as the α7 nicotinic-receptor.

Hence, in a second aspect, there is provided a receptor antagonistcomprising a cyclic polypeptide, derivative or analogue thereof.

Furthermore, in a third aspect, there is provided a cyclic polypeptide,derivative or analogue thereof, for use as a receptor antagonist.

As discussed above, the inventors have surprisingly shown that thecyclic polypeptides of the invention act as an inert allostericmodulator of the α7 nicotinic-receptor which antagonises the action ofT30 and amyloid beta peptides.

Hence, in a fourth aspect, there is provided a receptor allostericmodulator comprising a cyclic polypeptide, derivative or analoguethereof.

In a fifth aspect, there is provided a cyclic polypeptide, derivative oranalogue thereof, for use as a receptor allosteric modulator.

The cyclic polypeptide, derivative or analogue thereof is preferably thepolypeptide, derivative or analogue thereof according to the firstaspect. The receptor, which the cyclic polypeptide, derivative oranalogue thereof agent antagonises or allosterically modulates, may beany receptor, but is preferably an α7 receptor. Preferably, however, thereceptor, which the cyclic polypeptide, derivative or analogue thereofantagonises or allosterically modulates, is the α7 nicotinic-receptor.It is preferred that the cyclic polypeptide, derivative or analoguethereof antagonises or modulates an allosteric site on the receptor, andpreferably an Ivermectin-sensitive allosteric site of the receptor.

The inventors believe that they are the first to have shown that acyclic polypeptide can be used in therapy, for example in the treatmentof neurodegenerative disorders, such as Alzheimer's disease.

Thus, in a further aspect, there is provided a cyclic polypeptide,derivative or analogue thereof, for use in therapy or diagnosis.

In a further aspect, there is provided a cyclic polypeptide, derivativeor analogue thereof, for use in treating, ameliorating or preventing aneurodegenerative disorder.

In yet another aspect, there is provided a method of treating,ameliorating or preventing a neurodegenerative disorder in a subject,the method comprising, administering to a subject in need of suchtreatment, a therapeutically effective amount of a cyclic polypeptide,derivative or analogue thereof.

As discussed above, the inventor believes that the cyclic polypeptide,derivative or analogue thereof or the receptor antagonist or allostericmodulator described herein can be used to form the basis for treatingneurodegenerative disorders.

Thus, in a sixth aspect of the invention, there is provided the cyclicpolypeptide, derivative or analogue thereof according to the firstaspect, or the receptor antagonist according to the second aspect, orthe receptor allosteric modulator of the fourth aspect, for use intherapy or diagnosis.

In a seventh aspect, there is provided the cyclic polypeptide,derivative or analogue thereof according to the first aspect, or thereceptor antagonist according to the second aspect, or the receptorallosteric modulator of the fourth aspect, for use in treating,ameliorating or preventing a neurodegenerative disorder.

In a eighth aspect, there is provided a method of treating, amelioratingor preventing a neurodegenerative disorder in a subject, the methodcomprising, administering to a subject in need of such treatment, atherapeutically effective amount of the cyclic polypeptide, derivativeor analogue thereof according to the first aspect, or the receptorantagonist according to the second aspect, or the receptor allostericmodulator of the fourth aspect.

Preferably, the neurodegenerative disorder is selected from a groupconsisting of Alzheimer's disease; Parkinson's disease; Huntington'sdisease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type3; Amyotrophic Lateral Sclerosis (ALS); and Frontotemporal Dementia, andis preferably Alzheimer's disease.

The neurodegenerative disorder which is treated is preferably one eachis characterised by the damage or death of ‘Global’ neurons. Forexample, the neurodegenerative disorder may be selected from a groupconsisting of Alzheimer's disease; Parkinson's disease; Huntington'sdisease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type3; Amyotrophic Lateral Sclerosis (ALS); Frontotemporal Dementia; andSchizophrenia.

Preferably, the neurodegenerative disorder, which is treated, isAlzheimer's disease, Parkinson's disease, or Motor Neurone disease. Mostpreferably, the neurodegenerative disorder, which is treated, isAlzheimer's disease.

It will be appreciated that the cyclic polypeptide or receptorantagonist or the receptor allosteric modulator according to theinvention may be used in a medicament which may be used in a monotherapy(i.e. use of the cyclic polypeptide, derivative or analogue thereof),for treating, ameliorating, or preventing neurodegenerative disorder,such as Alzheimer's disease. Alternatively, the cyclic polypeptide orreceptor antagonist or the receptor allosteric modulator according tothe invention may be used as an adjunct to, or in combination with,known therapies for treating, ameliorating, or preventing Alzheimer'sdisease, such as other acetylcholinesterase inhibitors.

The cyclic polypeptide according to the invention may be combined incompositions having a number of different forms depending, inparticular, on the manner in which the composition is to be used. Thus,for example, the composition may be in the form of a powder, tablet,capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray,micellar solution, transdermal patch, liposome suspension or any othersuitable form that may be administered to a person or animal in need oftreatment. It will be appreciated that the vehicle of medicamentsaccording to the invention should be one which is well-tolerated by thesubject to whom it is given, and preferably enables delivery of thecyclic polypeptide across the blood-brain barrier.

It will be appreciated that the efficiency of any treatment for braindisorders depends on the ability of the candidate therapeutic compoundto cross the blood-brain barrier (BBB). The inventor believes thatpeptides of the size of Cyclic T14 may not gain ready access followingoral administration. However, it is well-known that, during Alzheimer'sdisease, the blood-brain barrier increases in permeability that couldallow Cyclic T14 to reach the central nervous system, indeed ideallyonly at the sites of degeneration where it is needed, i.e. where the BBBis compromised.

Two main strategies may be applied to cross the BBB with a largemolecule, such as Cylic T14 (i.e. NBP-14), including: (1) use ofnanoparticules as transporters to specifically target the brain anddeliver the active compound. This method has successfully been used todeliver peptides, proteins and anticancer drugs deliver to the brain;(2) use of cargo peptides. The addition of such a peptide specificallytransported across the BBB allows the transfer of the cyclic peptidethrough a facilitated manner.

Medicaments comprising cyclic polypeptides according to the inventionmay be used in a number of ways. For instance, oral administration maybe required, in which case the cyclic polypeptide may be containedwithin a composition that may, for example, be ingested orally in theform of a tablet, capsule or liquid. An alternative option foradministrating Cyclic T14 (i.e. NBP14) would be to use a nasal spray,since peptide administration by nasal spray reaches the brain faster andmore efficiently than oral or intravenous ways of administration (seehttp://memoryzine.com/2010/07/26/nose-sprays-cross-blood-brain-barrier-faster-and-safer/).Hence, compositions comprising cyclic polypeptides of the invention maybe administered by inhalation (e.g. intranasally). Compositions may alsobe formulated for topical use. For instance, creams or ointments may beapplied to the skin, for example, adjacent the brain.

Cyclic polypeptides according to the invention may also be incorporatedwithin a slow- or delayed-release device. Such devices may, for example,be inserted on or under the skin, and the medicament may be releasedover weeks or even months. The device may be located at least adjacentthe treatment site, e.g. the head. Such devices may be particularlyadvantageous when long-term treatment with cyclic polypeptides usedaccording to the invention is required and which would normally requirefrequent administration (e.g. at least daily injection).

In a preferred embodiment, medicaments according to the invention may beadministered to a subject by injection into the blood stream or directlyinto a site requiring treatment. For example, the medicament may beinjected at least adjacent the brain. Injections may be intravenous(bolus or infusion) or subcutaneous (bolus or infusion), or intradermal(bolus or infusion).

It will be appreciated that the amount of the cyclic polypeptide that isrequired is determined by its biological activity and bioavailability,which in turn depends on the mode of administration, the physiochemicalproperties of the cyclic polypeptide and whether it is being used as amonotherapy or in a combined therapy. The frequency of administrationwill also be influenced by the half-life of the cyclic polypeptidewithin the subject being treated. Optimal dosages to be administered maybe determined by those skilled in the art, and will vary with theparticular cyclic polypeptide in use, the strength of the pharmaceuticalcomposition, the mode of administration, and the advancement of theneurodegenerative disease. Additional factors depending on theparticular subject being treated will result in a need to adjustdosages, including subject age, weight, gender, diet, and time ofadministration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10mg/kg of body weight of the cyclic polypeptide according to theinvention may be used for treating, ameliorating, or preventingneurodegenerative disease, depending upon which cyclic polypeptide isused. More preferably, the daily dose is between 0.01 μg/kg of bodyweight and 1 mg/kg of body weight, and most preferably betweenapproximately 0.1 μg/kg and 10 μg/kg body weight.

The cyclic polypeptide may be administered before, during or after onsetof neurodegenerative disease. Daily doses may be given as a singleadministration (e.g. a single daily injection or inhalation of a nasalspray). Alternatively, the cyclic polypeptide may require administrationtwice or more times during a day. As an example, cyclic polypeptides maybe administered as two (or more depending upon the severity of theneurodegenerative disease being treated) daily doses of between 0.07 μgand 700 mg (i.e. assuming a body weight of 70 kg). A patient receivingtreatment may take a first dose upon waking and then a second dose inthe evening (if on a two dose regime) or at 3- or 4-hourly intervalsthereafter. Alternatively, a slow release device may be used to provideoptimal doses of cyclic polypeptide according to the invention to apatient without the need to administer repeated doses.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g. in vivo experimentation, clinical trials,etc.), may be used to form specific formulations of the cyclicpolypeptide according to the invention and precise therapeutic regimes(such as daily doses of the agents and the frequency of administration).The inventor believes that she is the first to suggest ananti-neurodegenerative disease composition, based on the use of a cyclicpolypeptide of the invention.

Hence, in a ninth aspect of the invention, there is provided apharmaceutical composition comprising a therapeutically effective amountof the cyclic polypeptide, derivative or analogue thereof according tothe first aspect or the receptor antagonist according to the secondaspect or the receptor allosteric modulator of the fourth aspect, andoptionally a pharmaceutically acceptable vehicle.

The pharmaceutical composition is preferably an anti-neurodegenerativedisease composition, i.e. a pharmaceutical formulation used in thetherapeutic amelioration, prevention or treatment of a neurodegenerativedisorder in a subject, such as Alzheimer's disease.

The invention also provides in a tenth aspect, a process for making thepharmaceutical composition according to the ninth aspect, the processcomprising combining a therapeutically effective amount of the cyclicpolypeptide, derivative or analogue thereof according to the firstaspect or the receptor antagonist according to the second aspect or thereceptor allosteric modulator of the fourth aspect, with apharmaceutically acceptable vehicle.

The cyclic polypeptide, derivative or analogue thereof is preferablyCyclic T14 (i.e. NBP14) as disclosed herein, i.e. SEQ ID No:4.

A “subject” may be a vertebrate, mammal, or domestic animal. Hence,medicaments according to the invention may be used to treat any mammal,for example livestock (e.g. a horse), pets, or may be used in otherveterinary applications. Most preferably, however, the subject is ahuman being.

A “therapeutically effective amount” of cyclic polypeptide is any amountwhich, when administered to a subject, is the amount of active agentthat is needed to treat the neurodegenerative disorder condition, orproduce the desired effect.

For example, the therapeutically effective amount of cyclic polypeptideused may be from about 0.001 mg to about 800 mg, and preferably fromabout 0.01 mg to about 500 mg. It is preferred that the amount of cyclicpolypeptide is an amount from about 0.1 mg to about 100 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is anyknown compound or combination of known compounds that are known to thoseskilled in the art to be useful in formulating pharmaceuticalcompositions.

In one embodiment, the pharmaceutically acceptable vehicle may be asolid, and the composition may be in the form of a powder or tablet. Asolid pharmaceutically acceptable vehicle may include one or moresubstances which may also act as flavouring agents, lubricants,solubilisers, suspending agents, dyes, fillers, glidants, compressionaids, inert binders, sweeteners, preservatives, coatings, ortablet-disintegrating agents. The vehicle may also be an encapsulatingmaterial. In powders, the vehicle is a finely divided solid that is inadmixture with the finely divided active agents according to theinvention. In tablets, the active agent (i.e. the modulator) may bemixed with a vehicle having the necessary compression properties insuitable proportions and compacted in the shape and size desired. Thepowders and tablets preferably contain up to 99% of the active agents.Suitable solid vehicles include, for example calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, polyvinylpyrrolidine, low melting waxes and ion exchangeresins. In another embodiment, the pharmaceutical vehicle may be a geland the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and thepharmaceutical composition is in the form of a solution. Liquid vehiclesare used in preparing solutions, suspensions, emulsions, syrups, elixirsand pressurized compositions. The active agent according to theinvention (the cyclic polypeptide) may be dissolved or suspended in apharmaceutically acceptable liquid vehicle such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid vehicle can contain other suitable pharmaceutical additivessuch as solubilisers, emulsifiers, buffers, preservatives, sweeteners,flavouring agents, suspending agents, thickening agents, colours,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid vehicles for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the vehicle can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile liquid vehiclesare useful in sterile liquid form compositions for parenteraladministration. The liquid vehicle for pressurized compositions can be ahalogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions orsuspensions, can be utilized by, for example, intramuscular,intrathecal, epidural, intraperitoneal, intravenous and particularlysubcutaneous injection. The cyclic polypeptide may be prepared as asterile solid composition that may be dissolved or suspended at the timeof administration using sterile water, saline, or other appropriatesterile injectable medium.

The cyclic polypeptide and compositions of the invention may beadministered orally in the form of a sterile solution or suspensioncontaining other solutes or suspending agents (for example, enoughsaline or glucose to make the solution isotonic), bile salts, acacia,gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitoland its anhydrides copolymerized with ethylene oxide) and the like. Thecyclic polypeptide used according to the invention can also beadministered orally either in liquid or solid composition form.Compositions suitable for oral administration include solid forms, suchas pills, capsules, granules, tablets, and powders, and liquid forms,such as solutions, syrups, elixirs, and suspensions. Forms useful forparenteral administration include sterile solutions, emulsions, andsuspensions.

Although the inventors have demonstrated the surprising therapeuticeffects of the cyclic polypeptide, derivative or analogue thereofaccording to the first aspect, due to its antagonistic nature, theybelieve that it will also be useful in non-clinically relatedexperiments designed to investigate the structure and/or function of theα7 nicotinic-receptor.

Hence, in a further aspect, there is provided use of the cyclicpolypeptide, derivative or analogue thereof according to the firstaspect, in an in vitro or ex vivo analytical method for investigating α7nicotinic-receptor.

Preferably, the method comprises investigating the allosteric site ofthe α7 nicotinic-receptor. Preferably, the method comprises using thecyclic peptide to prevent additional influx of calcium through the α7nicotinic-receptor. The cyclic peptide preferably acts as an antagonistand blocks the calcium ions.

It will be appreciated that the invention extends to any nucleic acid orpeptide or variant, derivative or analogue thereof, which comprisessubstantially the amino acid or nucleic acid sequences of any of thesequences referred to herein, including functional variants orfunctional fragments thereof. The terms “substantially the aminoacid/nucleotide/peptide sequence”, “functional variant” and “functionalfragment”, can be a sequence that has at least 40% sequence identitywith the amino acid/nucleotide/peptide sequences of any one of thesequences referred to herein, for example 40% identity with the sequenceidentified as SEQ ID No:1-6, and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identitywhich is greater than 65%, more preferably greater than 70%, even morepreferably greater than 75%, and still more preferably greater than 80%sequence identity to any of the sequences referred to are alsoenvisaged. Preferably, the amino acid/polynucleotide/polypeptidesequence has at least 85% identity with any of the sequences referredto, more preferably at least 90% identity, even more preferably at least92% identity, even more preferably at least 95% identity, even morepreferably at least 97% identity, even more preferably at least 98%identity and, most preferably at least 99% identity with any of thesequences referred to herein.

The skilled technician will appreciate how to calculate the percentageidentity between two amino acid/polynucleotide/polypeptide sequences. Inorder to calculate the percentage identity between two aminoacid/polynucleotide/polypeptide sequences, an alignment of the twosequences must first be prepared, followed by calculation of thesequence identity value. The percentage identity for two sequences maytake different values depending on:—(i) the method used to align thesequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman(implemented in different programs), or structural alignment from 3Dcomparison; and (ii) the parameters used by the alignment method, forexample, local vs global alignment, the pair-score matrix used (e.g.BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional formand constants.

Having made the alignment, there are many different ways of calculatingpercentage identity between the two sequences. For example, one maydivide the number of identities by: (i) the length of shortest sequence;(ii) the length of alignment; (iii) the mean length of sequence; (iv)the number of non-gap positions; or (iv) the number of equivalencedpositions excluding overhangs. Furthermore, it will be appreciated thatpercentage identity is also strongly length dependent. Therefore, theshorter a pair of sequences is, the higher the sequence identity one mayexpect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein orDNA sequences is a complex process. The popular multiple alignmentprogram ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22,4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882)is a preferred way for generating multiple alignments of proteins or DNAin accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty=15.0, Gap ExtensionPenalty=6.66, and Matrix=Identity. For protein alignments: Gap OpenPenalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA andProtein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the artwill be aware that it may be necessary to vary these and otherparameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two aminoacid/polynucleotide/polypeptide sequences may then be calculated fromsuch an alignment as (N/T)*100, where N is the number of positions atwhich the sequences share an identical residue, and T is the totalnumber of positions compared including gaps but excluding overhangs.Hence, a most preferred method for calculating percentage identitybetween two sequences comprises (i) preparing a sequence alignment usingthe ClustalW program using a suitable set of parameters, for example, asset out above; and (ii) inserting the values of N and T into thefollowing formula:—

Sequence Identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known tothose skilled in the art. For example, a substantially similarnucleotide sequence will be encoded by a sequence which hybridizes toDNA sequences or their complements under stringent conditions. Bystringent conditions, we mean the nucleotide hybridises to filter-boundDNA or RNA in 3× sodium chloride/sodium citrate (SSC) at approximately45° C. followed by at least one wash in 0.2×SSC/0.1% SDS atapproximately 20-65° C. Alternatively, a substantially similarpolypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100amino acids from the sequences shown in SEQ ID No: 1-6.

Due to the degeneracy of the genetic code, it is clear that any nucleicacid sequence described herein could be varied or changed withoutsubstantially affecting the sequence of the protein encoded thereby, toprovide a functional variant thereof. Suitable nucleotide variants arethose having a sequence altered by the substitution of different codonsthat encode the same amino acid within the sequence, thus producing asilent change. Other suitable variants are those having homologousnucleotide sequences but comprising all, or portions of, sequence, whichare altered by the substitution of different codons that encode an aminoacid with a side chain of similar biophysical properties to the aminoacid it substitutes, to produce a conservative change. For example smallnon-polar, hydrophobic amino acids include glycine, alanine, leucine,isoleucine, valine, proline, and methionine. Large non-polar,hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.The polar neutral amino acids include serine, threonine, cysteine,asparagine and glutamine. The positively charged (basic) amino acidsinclude lysine, arginine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. It will thereforebe appreciated which amino acids may be replaced with an amino acidhaving similar biophysical properties, and the skilled technician willknow the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made, byway of example, to the accompanying Figures, in which:—

FIG. 1 is a bar chart showing no effect of Cyclic T14 alone on PC12 cellviability after 1 hour treatment. Data represent mean±SEM, N=6;

FIG. 2 is a graph showing no change in enzymatic activity of AChE(determined by its ability to cleave increasing concentrations of asubstrate) in the presence or absence of Cyclic T14. In contrast,Galanthamine displays highly significant competitive inhibition ofenzyme activity. Data represent mean±SD, N=4;

FIG. 3 is a bar chart showing the non-specific toxic effect of H₂O₂ (100μM) alone, and its persistence when combined with Cyclic T14 (100 nM) oncell viability. Data represent mean±SEM, N=6. * vs Control; *P<0.05,***P<0.01;

FIG. 4 shows the effect after 1 hour treatment of (A) β-Amyloid (10 μM),(B) T14 (10 μM) and (C) T30 (10 μM) alone, and combined at time zerowith Cyclic T14 (100 nM). Data represent mean±SEM, N=12. * vs Control;*P<0.05, ***P<0.001;

FIG. 5 is a bar chart showing AChE activity after treatment of PC12cells with T30 (10 μM) alone, T14 (10 μM) alone or combined with CyclicT14 (100 nM). Data represent mean±SEM, N=6. * vs Control, P<0.001; #within groups, ^(###)P<0.001;

FIG. 6 shows competition curves of the inhibition of [³H] Ivermectinbinding by β-Amyloid, T30 and T14 in membranes of PC12 cells, as alsoshown in rat brain membranes. Data represent the means±SEM, N=3;

FIG. 7 shows competition curves of the inhibition of [³H] Ivermectinbinding by Cyclic T14 and Galanthamine in membranes of PC12 cells, asalso shown in rat brain membranes. Data represent the means±SEM, N=6;

FIG. 8 is a bar chart showing the minimum effective concentration ofGalanthamine (100 nM) and Cyclic T14 nM) against β-Amyloid (10 μM) oncell viability. Data represents mean±SEM, N=6. * vs Control; *P<0.05;

FIG. 9 is a graph showing the dose response effect of Cyclic T14 (1 nM)against β-Amyloid (10 μM) on cell viability. Data represents mean±SEM,N=6. * vs Control; *P<0.05;

FIG. 10 is a bar graph showing the intracellular levels of Ca²⁺ in ratbrain slices after 2 hours of different treatments. Data representsmean±SEM, N=4;

FIG. 11 shows the amino acid sequence alignment of the peptides, T30,T15, T14 and β-amyloid (Aβ)

FIG. 12 is a diagram showing the binding sites of β-amyloid and T30 onthe α7-nAChR;

FIG. 13 is a graph showing the protective effect of differentconcentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) onCalcium influx induced by T30. The values were fitted to a non linearcurve with the logarithm of the inhibitor concentrations, NBP-14, versusthe response of the T30, by using GraphPad Prism;

FIG. 14 is a graph showing the protective effect of differentconcentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) onAChE release induced by T30. The values were fitted to a non-linearcurve with the logarithm of the inhibitor concentrations, NBP-14, versusthe response of the T30, by using GraphPad Prism;

FIG. 15 is a graph showing the protective effect of differentconcentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) oncell viability induced by T30. The values were fitted to a non linearcurve with the logarithm of the inhibitor concentrations, NBP-14, versusthe response of the T30, by using GraphPad Prism;

FIG. 16 is a graph showing overall cortical response to thalamicelectrical stimulation under different T30 concentrations. (A) Synchronyof neuronal population activity, measured as fractional change influorescence intensity. (B) Spread of population activity, measured asthe number of active pixels—defined as pixels showing more than 20% ofthe max intensity given off by any single pixel within the Region ofInterest;

FIG. 17 shows isolation and linear analysis of the rise phase of thespread of assemblies under different T30 concentrations, as seen fromFIG. 16. Increasing concentrations of T30 show a dose-dependent decreasein the linear slope, equivalent to the velocity of propagation of theassembly;

FIG. 18 shows analysis of spread dynamics of thalamo-cortically-evokedneuronal assemblies under T30 and NBP-14 treatment, n=3. Analysis of therise phase (left panel) shows a latent effect of T30 in increasing theslope (velocity) of propagation. The slope shows significant increasesonly about 45-60 minutes after initial T30 perfusion (yellow bar,effects become apparent during orange perfusion—with perfusion of 5 nMNBP-14). This sharp increase trend is slowed down during the 100 nMNBP-14 perfusion (red bar), and then reversed back to baseline levelsduring the final 300 nM NBP-14 perfusion (black bar). Plateau phaseanalysis (right panel) shows a similar profile of effects. The top panelshows a box and whisker plot averaging the behaviour of the assemblies'spread under the different drug treatments: a similar trend as in therise-phase slope analysis can be seen, and though non-significant, thetrend remains that T30 gradually increases the spread of evokedassemblies until a sufficiently high NBP-14 concentration reaches therecording bath (100 nM) where this excitatory trend is reversed backtowards control levels (blue) at 300 nM NBP-14 perfusion (black);

FIG. 19 shows qualitative results from the three experiments (ie left,centre and right-hand columns) where T30 effects were tested againstincreasing concentrations of NBP-14 (0.1, 5, 100 & 300 nM). Top panelshows the two main averaged-data graphs: left—Intensity of fluorescencesignal, and right—spread of evoked assemblies. Bottom panel shows‘space-time’ maps mapping the activity of a row of pixels lying over thearea of interest (y-axis) over time (310 ms total, x-axis) for eachperfusion conditions. The drastic reduction in fluorescence intensity asa result of T30 and NBP-14 co-perfusion is clearly evident, as it is onthe Intensity graph. Note: the space-time maps are labelled with theirrespective perfusion (left), and are colour-coded to their correspondingtraces both in the intensity (right) and spread (left) graphs;

FIG. 20 shows a schematic of the procedure followed during the in vivotesting of NBP-14. The day before the surgery all animals are tested inorder to reveal any impairment. The day of the surgery is considered asday 0 of the study. On day one a paw placement test allowed theselection of the 16 out of 24 best subjects in order to inject with thevehicle or NPB-14. On day 2 a paw placement test was performed;

FIG. 21 shows the effect of NBP-14 against 6-OHDA in comparison withBaseline and 6OHDA alone in the Paw placement test. 6-OHDA **P<0.01 and6OHDA+NBP14 ***P<0.001 vs Baseline. (N=8);

FIG. 22 shows the cascade of events resulting from the effect of T30 ina cell;

FIG. 23 shows full-length APP is reduced by NBP-14. Data representexpression of APP in solubilized PC12 cells after 3 different 1 hourtreatments. Data are represented as mean±SEM, n=2; and

FIG. 24 shows immunodetection by western blot represented in the graph.The 3 different treatments show different levels of expression of APP(values represented in FIG. 24. For each condition protein is correctedby levels of GAPDH.

FIG. 25 shows that T30 induces release of Aβ42, an effect reversed byNBP14. Graph showing the release of Aβ42 in control conditions, inpresence of T30 and in presence of T30 and NBP-14. The results arerepresented as mean±SEM (n=4).

EXAMPLES

It should be noted that SEQ ID No:4 is referred to herein as “cyclatedT14”, “CT14” or “NBP14”.

Materials and Methods

Cyclisation of Peptides

Three techniques were used to achieve cyclization of linear peptidesdescribed herein, i.e. sidechain-to-sidechain, sidechain-to-backbone,and head-to-tail (C-terminus to N-terminus) cyclization. Head-to-tailcyclization has been investigated extensively, and can involve directedCys-Cys disulphide cyclization (up to two per molecule). Carefulmonitoring of the reaction ensures 100% cyclization. Two generalapproaches are used for synthesis: (1) classical solution-phase linearpeptide cyclization under high dilution conditions; and (2) resin-basedcyclization. Two distinct protocols were employed in the solid phasesynthesis (1):—

(a) The on-resin cyclization of a peptide anchored via a sidechainfunctional group, such as imidazole, 3 acid,4 amine′ or alcohol, wascarried out. The peptide was orthogonally protected as an ester at theC-terminus, and the peptide was then assembled through regular Boc orFmoc synthesis followed by saponification, cyclization and cleavage.

(b) Another protocol that was used was the cyclization cleavageapproach, in which the cyclic peptide was synthesized by cyclizationafter step-wise linear peptide synthesis. One advantage of this methodis that the sidechain does not need to be anchored, making the approachmore general than (a). (Christopher J. White and Andrei K. Yudin (2011)Nature Chemistry 3; Valero et al (1999) J Peptide Res. 53, 76-67; LihuYang and Greg Morriello (1999) Tetrahedron Letters 40, 8197-8200;Parvesh Wadhwani et al (2006) J. Org. Chem. 71, 55-61).

Resultant samples of cyclic peptides were analysed by MALDI-TOF MS.

PC12 Cell Culture

PC12 cells are a cloned, pheochromocytoma cell line derived from theadrenal medulla (Greene and Tischler, 1976, Proc Natl Acad Sci USA 73:2424-2428; Mizrachi et al., 1990, Proc Natl Acad Sci USA 87: 6161-6165).They are easily cultured and readily accessible to experimentalmanipulations. Since chromaffin cells are derived from the neural crestbut are located in the centre of an accessible peripheral organ (theadrenal medulla) they have been described as offering a ‘window’ intothe brain (Bornstein et al., 2012, Mol Psychiatry 17: 354-358). Thesecells serve as a powerful, albeit novel, in vitro model for studying thestill unknown primary process of neurodegeneration and the reasons whythey are useful for this project are the following: the adrenal medullain Alzheimer's patients shows various pathological features reminiscentof those seen in the CNS, e.g. numerous Lewy-body like inclusions,neurofibrillary tangles and paired helical filaments, as well asexpression of amyloid precursor protein (APP) (Takeda et al., 1994,Neurosci Lett 168: 57-60). Moreover Appleyard and Macdonald (1991,Lancet 338: 1085-1086) demonstrated a selective reduction only in thesoluble i.e. releasable form of AChE from the adrenal gland in AD,perhaps due to its enhanced secretion into the plasma, where it iselevated in AD patients (Atack et al., 1985, J Neurol Sci 70: 1-12;Berson et al., 2008, Brain 131: 109-119).

Wild-type PC12 cell were provided by Sigma-Aldrich (St. Louis, Mo.). Theculture was routinely plated in 100 mm dishes (Corning) coated withcollagen (2 μg/cm²) and maintained in growth medium with MinimumEssential Medium Eagle (MEM) supplemented with heat-inactivated 10%horse serum (HS) and 5% foetal bovine serum (FBS), 10 mM HEPES, 2 mML-Glutamine and 1:400 Penicillin/streptomycin solution. Cells weremaintained at 37° C. in a humidified atmosphere 5% CO₂ and the mediumwas replaced every 2 days. For splitting, cells were dislodged from thedish using a pipette with medium, with a portion of these replated ontonew cultured dishes. Cells were used between passages 12 and 25.

Cell Membrane Preparation

PC12 membranes where obtained to perform binding assays. PC12 cells weregrown until confluence on 100 mm plates. Growth medium was removed andice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 4.5 μg/μl aprotininand 0.1 mM phenylmethylsulphonylfluoride (PMSF) were added. Cells weremechanically detached and pelleted by centrifugation (1040×g) for 4minutes at 4° C. Pellets were homogenized with a Polytron andcentrifuged (13000×g) for 20 minutes at 4° C. The pellets wereresuspended in fresh buffer and incubated at 37° C. for 10 minutes toremove endogenous neurotransmitters. The samples were subsequentlyre-centrifuged. The final pellet was resuspended in buffer and theprotein concentration determined using the Bradford Reagent(Sigma-Aldrich, St. Louis, Mo.). The cell membrane preparation wasstored at −80° C.

β-Amyloid Preparation

β-Amyloid (1-42) fibrils were prepared as described by provider (Abcam,Cambridge UK)). 1 mg of β-Amyloid (1-42) was dissolved in 212 μl of1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 10 μl of NH₄OH. Aftersonication and distribution of 10 μl of sample per tube, samples weredried in a speed vacuum drier (Thermo Fisher Scientific, Loughborough,UK) and stored at −20° C. For experiments, samples were diluted in 2 μlof DMSO (5 mM) and 98 μl of HCl (0.01N) to ensure fibril formation andincubated over night at 37° C.

[³H] Ivermectin Binding Assay

For the binding with PC12 membranes, each incubation was performed inpolystyrene tube (VWR International Ltd; Leicestershire, UK) containing0.25 ml of membranes diluted in Tris-HCl 50 mM buffer (containing 50 μgof PC12 membranes) with 5 nM [³H] Ivermectin (American RadiolabeledChemicals, USA) in the absence or presence of different concentrationsof AChE peptide T30, β-Amyloid or Cyclic T14 (0.1, 0.5, 0.7, 1, 2, 10μM) diluted in Tris-HCl 50 mM, in a final volume of 0.5 ml for 2 h at 4°C.

Thereafter, samples were filtered through Brandel GB glass fibre filters(MD, USA); pre-soaked in 0.5% polyethylenemine by a Harvester (Brandel;MD, USA). Tubes were washed 3 times with ice cold 50 mM Tris-HCl buffer.Radioactivity in the tubes was counted by scintillation spectrometryusing a 300SL Liquid scintillation counter (Lablogic Systems Limited,UK). Specific binding was determined by subtracting the non-specific(cells treated with Ivermectin 30 μM) value to all the tubes.

Cell Viability Assay

The cell viability assay used was the sulforhodamine B (SRB)colorimetric assay for toxicity screening. The day before of theexperiment cells were seeded onto collagen-coated 96-well plates in aconcentration of 40,000 cells/well. Cell concentration was determined bythe Fuchs-Rosenthal chamber. Drugs were prepared in MEM containingL-Glutamine and cells were treated with different concentrations ofCyclic T14 (0.1-100 μM) and T30, T14 and Aβ (10 μM) alone or combinedwith Cyclic T14 (0.1 and 0.7 μM). After treatment, medium was replacedand cells were fixed by adding 100 μl of 10% Trichloroacetic Acid (TCA)for 1 h at 4° C. Thereafter, cells were washed with H₂O and stained with100 μl of a 0.057% SRB solution in 1% Acetic acid (HAc) for 30 minutesat room temperature. After staining cells were washed with 1% HAc forremoving the excess of SRB and then incubated with 200 μl of 10 mM Trisbase (pH 10.5) and shake it for 5 minutes to solubilise theprotein-bound dye. Measurement of the absorbance took place in a V_(Max)Kinetic Microplate Reader (Molecular Devices) at 490 nm.

Acetylcholinesterase Activity Assay

AChE activity was measured using the Ellman reagent that measures thepresence of thiol groups as a result of AChE activity. Cells were platedthe day before the experiment as for the cell viability assay. Cellswere treated with different concentrations of Cyclic T14 (0.1-100 μM)and T30, T14 and AP 10 μM alone or combined with Cyclic T14 (0.1 and 0.7μM). After treatment, supernatant (perfusate) of each treatment wascollected and 25 μL of each condition were added to a new flat bottomed96 well plate followed by the addition of 175 μL of Ellman reagent(Solution A: KH₂PO₄ 139 mM and K₂HPO₄ 79.66 mM, pH 7.0; solution B(substrate): Acetylthiocholine Iodide 11.5 mM; Solution C (Reagent):5,5′-Dithiobis (2-nitrobenzoic acid) 8 mM and NaHCO₃ 15 mM). The Ellmanreagent was prepared as a mixture of the 3 solutions in a ratio33(A):3(B):4(C). Absorbance measurements were taken at regular intervals(3, 10, 30 and 60 mins) across experiments at 405 nm.

Calcium Fluorometry

Increases in intracellular Ca²⁺ were monitored by measuring changes influorescence in cells loaded with Fluo-4 (Life Technologies Corporation,UK). The brain slices were incubated for 2 hours in 124 mM NaCl, 3.7 mMKCl, 26 mM NaHCO₃, 2 mM CaCl₂, 1.3 mM MgSO₄, 1.3 mM KH₂PO₄ and 10 mMglucose; pH: 7.1 containing β-Amyloid, Cyclic T14 or β-Amyloid+CyclicT14. After the 2 hours, slices were incubated in the dark for 40 minutesat room temperature with 1.2 ml/well of loading medium that contained:Tyrode's salt solution (TSS; 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 2.5mM CaCl₂, 0.2 mM NaH₂PO₄, 12.0 NaHCO₃, 5.5 glucose, pH 7.4), Fluo-4 (2μM), Pluronic F127 (0.02%) and probenecid (2 mM). Probenecid is ablocker of the multidrug resistant protein, an ion transporter, andavoids the excretion of the fluorescent molecule from the cell. Afterincubation, slices were washed with TSS and 1200 μl/well ofde-esterification medium, containing TSS and probenecid, were added.Slices were incubated in the dark for 20 minutes at 22° C. Fluorescencemeasurements (excitation 485 nm, emission 538 nm) were recorded in aFluostar Optima (BMG, UK) plate reader.

Drugs and Reagents

MEM, culture serums, antibiotics, collagen, sulforhodamine B, Ivermectinand buffers reagents were provided by Sigma-Aldrich (St. Louis, Mo.).T30, T14 AChE peptides and Cyclic T14 were synthesized by GenosphereBiotechnologies (France). Stocks of peptides were diluted in distilledwater.

Data Analysis

In each of the different techniques, the statistics analysis wasperformed with the average of the percentage values of 12 or moreexperiments. Comparisons between multiple treatment groups and the samecontrol were performed by one-way analysis of variance (ANOVA) andTukey's post-hoc tests using GraphPAD Instat (GraphPAD software, SanDiego, Calif.). These tests compare the means of every treatment to themeans of every other treatment; that is, apply simultaneously to the setof all pairwise comparisons and identify where the difference betweentwo means is greater than the standard error would be expected to allow.Statistical significance was taken at a P value <0.05. Graphs wereplotted using GraphPAD Prism 6 (GraphPAD software, San Diego, Calif.).In the case of the binding experiment, results were obtained as countsper minute (cpm) and transformed to percentages related to control.Results were fitted to a model of one site competition binding usingGraphPad Prism. In the case of the calcium results, the EC₅₀ values werecalculated by fitting the logarithm of the experimental data points to asingle site Hill equation using a non-linear regression curve usingGraphPad Prism.

Example 1 Cyclisation of T14

The inventor synthesised an agent that selectively targets theallosteric site on the α7 nicotinic acetylcholine receptor, to competefor binding with T14/T30 and also to antagonise β-amyloid. The agent isa cyclic form of T14 having the amino acid sequence: AEFHRWSSYWVHWK [SEQID No:4], with the N-terminal alanine residue being connected to theC-terminal lysine residue. Genosphere Biotechnologies (France) performedthe cyclisation of T14 by transforming the linear peptide into anN-terminal to C-terminal lactam. The following examples demonstrate forthe first time how the Cyclic T14 peptide blocks the established toxiceffects of the T30 peptide and amyloid in vitro.

Example 2 Cyclic T14 is not Toxic when Applied Alone

Using sulforhodamine B (SRB) as a cell viability detection method, PC12cells were treated for 1 hour with Cyclic T14 produced in example 1. Asa result, no changes in cell viability were observed suggesting notoxicity at concentrations as high as 100 μM (100 nM: 98.76±15.15; 700nM: 106.94±19.92; 1 μM: 104.82±10.9; 100 μM: 93.58±11.62) (see FIG. 1).

Example 3 Cyclic T14 does not Affect AChE Enzymatic Activity

The inventor next decided to confirm whether or not Cyclic T14 affectsthe enzymatic activity of acetylcholinesterase (AChE). AChE enzymaticactivity was measured using the acetylcholinesterase activity assay. Theinventor found that the presence of Cyclic T14 (2 μM) did not affectenzyme activity of acetylcholinesterase: in contrast Galanthamine (2 μM)was strongly inhibitory (see FIG. 2).

Example 4 Cyclic T14 does not Protect Against Non-Specific Toxicity ofHydrogen Peroxide

The inventors then determined whether or not Cyclic T14 protects PC 12cells against the non-specific cytotoxic effects of the hydrogenperoxide. As can be seen in FIG. 3, there is no significant differencewhen H₂O₂ is given alone or in combination with Cyclic T14.

Example 5 Cyclic T14 Protects Cells from T14, T30 and β-Amyloid Toxicity

Using SRB as a cell viability detection method, PC12 cells were treatedfor 1 hour with (4A)β-amyloid, (4B) linear T14, or (4C) T30, eitheralone or combined with Cyclic T14 (100 nM). As shown in FIG. 4, thethree peptides alone induce a decrease in cell viability (Aβ:690.875±4.38; T14: 83.02±5.385 and T30: 68.395±3.095), but when combinedwith Cyclic T14 cells were surprisingly protected from death (Aβ+C14:94.475±7.4; T14+C14: 99.4±12.475; T30+C14: 88.59±8.785).

Example 6 Cyclic T14 Blocks AChE Release Induced by T14 and T30

The colourmetric Ellman assay was used to assess AChE activity as acompensatory response after a toxic stimulus. Cells were treated for 1hour with linear T14 and T30 (10 μM) alone and combined with Cyclic T14(100 nM) (see FIG. 5). All the peptides induce an increase of AChEactivity (T30: 129.10±1.18; T14: 123.0±0.62) that was partially blockedwhen combined with the Cyclic T14 (T30+C14: 110.58±0.80; T14+C14:112.30±1.39).

Example 7 β-Amyloid, T30 and T14 Displace [3H] Ivermectin Binding

In order to demonstrate that the α7nAChR (the α7 nicotinic acetylcholinereceptor) is a target for the β-Amyloid, T30 and T14 in the preparationsused here. [³H] Ivermectin binding assays were performed on PC12 cellmembrane and demonstrate in a log dose-response manner a decrease of theaffinity of the allosteric site of the receptor where the ligand [³H]Ivermectin binds (see Table 1, FIG. 6).

TABLE 1 Data showing the percentage of [³H] Ivermectin binding on PC12cells in the presence of different concentrations of β-Amyloid. T30 andT14, N = 3. % [³H] Ivermectin (Mean ± SEM) β-Amyloid T30 T14 1 nM 100.00± 9.70  109.16 ± 11.9  100 ± 9.91 10 nM 73.61 ± 11.12  116.63 ± 13.2590.92 ± 2.38 100 nM 41.17 ± 8.90  106.15 ± 8.04 88.92 ± 3.82 1 μM 29.98± 12.20 97.41 ± 7.9 85.17 ± 3.03 5 μM 34.49 ± 17.29  80.22 ± 3.81 85.36± 3.96

Example 8 Cyclic T14 Displaces [3H] Ivermectin Binding with GreaterEfficacy than Galanthamine

Low micromolar concentrations of cyclic T14 displaced [³H] Ivermectinwith similar affinity but with significantly greater efficacy thanGalanthamine.

TABLE 2 Data showing the percentage of [³H] Ivermectin binding on PC12cells in the presence of different concentrations of Cyclic T14 andGalantamine, N = 6 % [³H] Ivermectin (Mean ± SEM) Cyclic T14Galanthamine 100 nM 98.10 ± 3.28 100.00 ± 11.48 200 nM 80.81 ± 4.3797.86 ± 1.40 500 nM 79.72 ± 6.76 90.96 ± 1.87 700 nM  62.26 ± 17.6369.68 ± 9.87 1 μM 29.006 ± 8.23  67.17 ± 6.64 2 μM  13.46 ± 10.40 66.32± 4.29

Example 9 Cyclic T14 Protects Cells from β-Amyloid Toxicity with GreaterEfficacy than Galanthamine

Using SRB as a cell viability detection method, PC12 cells were treatedfor 1 hour with β-amyloid either alone or combined with Cyclic T14 (1nM) or Galanthamine (100 nM). As shown in FIG. 8, Cyclic T14 protectedagainst Aβ toxicity (97.34±9.57) in a dose two orders of magnitude lowerthan Galanthamine (98.79±14.21).

Example 10 Minimum Concentration of Cyclic T14 Required for 100%Protection Against β-Amyloid

Using SRB as a cell viability detection method, PC12 cells were treatedfor 1 hour with β-amyloid combined with Cyclic T14 in concentrationsincreasing from 0.5 nM to 100 nM (0.5: 88.49±10; 1: 97.34±9.57; 10:102.28±8.53; 50: 101.79±13.99; 100: 103.68±6.34). The threshold dose forfull protection was 1 nM (FIG. 9).

Example 11 Cyclic T14 Reduces Ca²⁺ Levels in Rat Brain Slices

Fluorometry was used to detect variations in calcium levels aftertreatment for two hours with Cyclic T14 1 μM, β-Amyloid 10 μM and bothcombined. Cyclic T14 does not change the basal level of intracellularcalcium whilst β-Amyloid induces to increase the intracellular calciumlevel, which is returned to baseline by Cyclic T14 (see FIG. 10).

Example 12 T30 Exhibits a High Binding Affinity for the Allosteric Siteof the α7 Nicotinic-Receptor

Using tests for viability, the inventor has shown that T30 has a bindingaffinity approximately three orders of magnitude higher (5 nM) for theallosteric site on the α7 nicotinic-receptor, than drugs currently inclinical use, e.g. galanthamine (10 μM).

General Discussion

Cyclic T14 is a novel α7 nicotinic-receptor inert allosteric modulatorof the α7 nicotinic-receptor which antagonises the action of T30 andamyloid beta peptides Cyclic T14 is a novel α7 nicotinic-receptorantagonist. The inefficacy of protection against the non-specific agenthydrogen peroxide suggests that the blocking action of Cyclic T14 isselective and receptor mediated. Cyclic T14 antagonises the toxiceffects of T30 in a variety of tests indicating that it prevents theadditional influx of calcium through an allosteric site on the α7receptor by competing for binding with T30 as well as with amyloid. Theenhanced stability of cyclic peptides would account for this effectivedisplacement.

Why would a Cyclic T14-Based Drug be More Effective than CurrentlyAvailable Treatments?

The inventor has recently shown that T30 has a binding affinityapproximately three orders of magnitude higher (5 nM) for the allostericsite on the α7 receptor, than drugs currently in clinical use, e.g.galanthamine (10 μM). Indeed, this observation would suggest the reasonwhy such drugs currently being prescribed have proved relativelydisappointing (See Table 1; Kramp & Herrling, 2011, NeurodegenerativeDis 8, 44-94): if endogenous T30, in excess in the Alzheimer patient'sbrain, is already occupying the key site, it will not be displaced bylow-affinity competition. However, it would be blocked by an agent withvery similar or indeed superior binding affinities, as suggested here(see FIG. 7). Such an agent has therefore the potential for being ahighly effective drug.

A further advantage of the Cyclic T14 is that, unlike galanthamine,which is additionally an AChE inhibitor, it would have no otherbiological actions, other than to bind to the receptor. If, as theinventor's previous work suggests (Greenfield, 2013, Chem Biol Interact.203(3):543-6), T30 is indeed the pivotal signalling molecule inneurodegenerative diseases, then its antagonism would be combattingthese diseases at the most fundamental and specific level. In any event,the observation that this novel agent also antagonises amyloid would beof great clinical interest, where amyloid is implicated in thedegenerative process, irrespective of its precise role. It should benoted that whilst other therapeutic candidates targeting theavailability of 3-Amyloid (e.g. gamma secretase inhibitors) have beenineffective, this is the first instance, of the effective blockade ofamyloid toxicity.

TABLE 3 Comparison of features of Galanthamine vs Cyclic T14-based drugGalanthamine Dream Drug Cyclic T14 Inhibits AChE (side Does not affectAChE activity ✓ effects) Known action at various Specific action at α7receptor ✓ receptors Micromolar affinity Nanomolar affinity ✓ Blocksβ-Amyloid at high Blocks β-Amyloid at low ✓ doses (0.1 μM) doses1 nM)Low permeability CNS Should have high permeability CNS Highbioavailability Should have low peripheral periphery (Side effects asbioavailability diarrhoea) Post-symptomatic Pre-symptomatic

The inventor believes that the current results suggest that theconformation of Cyclic T14 allows it to bind to its specific target, α7nicotinic-receptor. Referring to FIG. 12, there is shown a schematicdiagram of the α7 nicotinic-receptor. The homomeric receptor containsfive identical α7 subunits, which are each symmetrically arranged arounda central pore through which ions, such as Na⁺ and Ca²⁺, pass when thereceptor is activated. Each α7 subunit contains an orthosteric bindingsite (i.e. the active site) and an allosteric binding site. Normalphysiological activation of the receptor is achieved by the binding of asingle acetylcholine molecule to the interface of two α7 subunits viaeach of their orthosteric sites. Other known ligands of the orthostericsite include (but are not limited to) choline and Methyllycaconitine(MLA). Ligands of the allosteric site include (but are not limited to)linear and cyclical T14, cyclical and linear T30, galantamine,ivermectin and PNU12.

As shown in FIG. 10, although not wishing to be bound by this theory,the inventor believes that β-amyloid (Aβ) is capable of either (i)simultaneously binding to both the orthosteric and the allostericbinding sites of the α7 nicotinic-receptor, or (ii) non-specificallybinding to one either of these sites. The inventor has found thatcyclical T14 acts as an antagonist at the allosteric site.

Drug Design

The inventor believes that it will be possible to use the particularconformation of Cyclic T14 to design a much smaller chemical compoundwhich nonetheless still mimics the three-dimensional form of Cyclic T14and is able to cross the blood-brain barrier more readily.

Example 13 Physico-Chemical Characterisation of Cyclic T-14 (i.e.Referred to as “Nbp14”) BACKGROUND

The solubility of a compound in aqueous and organic solutions stronglyaffects its ability to cross physiological barriers in the body, such asgastric or enteral. In the case of drugs targeting brain diseases, e.g.dementia, an additional barrier has to be crossed, the Blood-BrainBarrier. The partition coefficient, also known as Log P, evaluates theability of a compound to solubilize in water and organic solvent, whichcorrelates with the capacity of a compound to cross the differentbiological barriers.

Detailed Methods

Solvent Preparation

Saturation of the solvents was performed as follows. 1-octanol wasagitated in the presence of water for 24 h at room temperature. mQ waterwas agitated in presence of 1-octanol for 24 h at room temperature. Thenthe solutions were left to equilibrate overnight at room temperature.Saturated solvents were collected, using syringes and needles, andstored at room temperature until further use.

Shake-Tube Method

Saturated water and saturated i-octanol were placed in a glass tube inthe following ratios: Each tube contained the equivalent of 0.25 mg ofcyclic T14. All tubes were then mixed for 4 h at room temperature. Afteragitation the tubes were left at room temperature to equilibrate.

Standard Curve

The concentrations of cyclic T14 used for the standard curve were: 0.5mg·ml⁻¹, 0.25 mg·ml⁻¹, 0.13 mg·ml⁻¹, 0.066 mg·ml⁻¹, 0.033 mg·ml⁻¹ and0.016 mg/ml⁻¹. The absorbance of the standard curve was measured at 280nm.

Sample Analysis

Both fractions of each sample were collected separately using a syringewith needle. The absorbance of all fractions was measured at 280 nm andthe concentration of all the fraction was estimated based on thestandard curve. The partition coefficient of cyclic T14 was calculatedusing the following equation:

Log P=Log(Concentration in Octanol/Concentration in Water)

The results from each condition were averaged in order to obtain the LogP of cyclic T14.

Results and their Implications

The average Log P of cyclic T14 is −0.5899. A negative value for Log Pmeans that the compound is more likely to be hydrophilic. However, a LogP close to 0 corresponds to a compound with the ability to be soluble ina lipophilic environment as well. Hence, NBP14 can be formulated tocross the BBB.

Example 14 Effects of T30 and Cyclic T-14 (i.e. NBP-14) in PC12 Cells

To characterize further the protective effects of NBP-14 against T30toxicity, the inventors have determined the concentration-effect onthree in vitro systems ((A) Calcium influx; (B) AChE release; (C) Cellviability), as detailed in the Methods section below.

Methods

(A) Calcium Influx

PC12 cells are plated in 200 μl of complete growth medium the day beforethe experiment in 96 well plates. On the day of the experiment, theFluo-8 solution (Abcam) is prepared (as provider protocol).Subsequently, 100 μl of growth medium is removed and 100 μl of Fluo-8solution is added. Treatments with T30 and NBP-14 are added andincubated for 30 minutes in the incubator and 30 minutes roomtemperature.

After 1 hour, the plate is placed in the fluorescence plate reader(Fluostar). Before reading the fluorescence, acetylcholine (ACh) 100 μMis prepared and placed in the Fluostar injector. For each well, thereading will be formed by a basal fluorescence followed by acetylcholineinjection that will induce an increase of calcium via nicotinicreceptors. The effects of T30 and NBP-14 are then evaluated.

(B) AChE Release

The protocol used to detect changes in AChE activity is the same asdescribed previously.

(C) Cell Viability

A Cell Counting Kit-8 (CCK-8) was used as an improvement of the SRBtechnique used before. By utilizing the highly water-soluble tetrazoliumsalt WST-8, CCK-8 produces a water-soluble formazan dye upon reductionin the presence of an electron carrier. WST-8 is reduced bydehydrogenases in cells to give a yellow colored product (formazan),which is soluble in the tissue culture medium. The amount of theformazan dye generated by the activity of dehydrogenases in cells isdirectly proportional to the number of living cells. PC12 cells areplated in 200 μl of complete growth medium the day before the experimentin 96 well plates. Treatments with T30 and NBP-14 are added andincubated for 1 hour in the incubator.

Subsequently, 100 μl of growth medium is removed and 10 μl of CCK-8(Cell Counting Kit-8) solution is added. The plate is incubated for 2hours in the incubator and then placed in the absorbance plate reader.The absorbance must be measured at 450 nm.

(A) Calcium Influx

As stated previously, T30 is a positive allosteric modulator of the α7nicotinic receptor. Hence the primary agonist acetylcholine was used tobenchmark the control calcium influx as 100%). T30 (5 uM) enhanced thiseffect until 171.05%±6.21%; N=3. Increasing concentrations of NBP-14 (5,7, 9, 10, 20, 50, 70, 1000, 5000 nM) were subsequently added todetermine the antagonism of these T30-induced increases. The values are(respectively) (%): 134.2497±6.85, 120.8612±8.65, 113.9162±8.82,140.776±12.16, 115.83±7.67, 110.3213±13.21, 125.9596±0.1, 99.85±0.32,115.1942±9.84, 79.99±14.04. FIG. 13 shows that NBP-14 blocks T30 effectsin a concentration manner, being protective at low nanomolar range.

(B) AChE Release

As described above, PC12 cells respond to the toxic effect of T30 with a‘compensatory’ response, i.e. an increase in released AChE activity:169.45%±2.11%; N=3. The inventors determined the dose-dependent effectof NBP-14 against 5 uM T30. The results show (FIG. 14) that NBP-14protects from AChE compensatory effects at high nanomolarconcentrations. The values are (respectively) (%): 130.73±1.84,111.68±2.26, 92.78±0.99, 82.56±2.38, 68.90±0.92, 65.12±1.32, 61.04±0.97,79.43±1.69±1.24, 83.91±1.24, 89.55±1.25.

(C) Cell Viability

T30 (5 uM) induces a 25% (74.309%±2.87%; N=3) decrease of cell viabilitythat is progressively blocked by NBP-14 in a concentration-effect manner(FIG. 15). The values are (respectively) (%): 76.25±7.51, 67.04±4.35,76.04±4.22, 71.36±1.64, 79.02±10.22, 75.19±3.9, 62.43±3.01, 78.10±2.16,116.65±3.62, 107.79±5.10. NBP-14 protects from T30 induced cell death inthe high nanomolar range.

Example 15 Effects of T30 and Cyclic T-14 (i.e. NBP14) on In VitroCortical Networks in Rat Brain Slices

Background

The T30 peptide is a 30-amino acid segment of acetylcholinesterase(AChE), from which the T14 is also cleaved. Both induce the same effectsuggesting their active sequence is present on the T14, and in turnpresent on the T30. The research has already shown the bioactivity ofT14 on mammalian brain slices as a highly modulatory agent. The effectsof the T14 have been reported to modulate cortical networks, inducingboth excitation and inhibition at different concentrations: at lowconcentrations the peptide triggers enhanced calcium influx via thealpha-7 receptor, but high concentrations induce such excessive amountsof calcium that the channel inactivates (Badin et al., 2013; Bon andGreenfield, 2003), as well as triggering neuronal plasticity (Greenfieldet al., 2004).

In order to gain further understanding of the actions of T14/30 on wholecortical networks, the relatively recent technique of voltage-sensitivedye imaging (VSDI) was used in order to monitor the dynamics ofcollective neuronal population activity, ‘neuronal assemblies’, in brainslices on a temporal scale of milliseconds (ms, commensurate withphysiological events) and micrometres (μm). Such a technique exploitsthe sensitivity of specific lipophilic molecules containing afluorescent core to changes in electrical potentials (Tominaga et al.,2000). Due to their lipophilic nature, these dye molecules embedthemselves in cell membranes, and alter their fluorescence reading withregards to the voltage potential across that specific membrane, whichare captured with a millisecond-resolution high-speed camera. As aresult, imaging using voltage-sensitive dyes provides a direct andon-line readout of electrical potential changes across neuronal cellmembranes with an unparalleled spatio-temporal resolution.

Using this technique the inventors can obtain comprehensive sets of dataon neuronal population activity such as (a) the intensity of theresponse in any given area, from which (b) the spread of elicitedneuronal assemblies can be measured, and from this parameter (c) thevelocity of propagation of the activity wave-front from the point ofinitiation (d) measured as the slope of the spread. Each of theseparameters have been measured independently for two experiments carriedout so far: 1) an investigation of the effects induced by increasingconcentrations (0.5, 0.75, 1 & 5 μM) of T30 on cortical populationactivity and responsiveness, and 2) assessing the antagonistic effectsof NBP-14 on a single, relatively high (1 uM), T30 concentration.

-   -   Technique used: voltage-sensitive dye imaging (Di-4-ANEPPS) of        thalamocortical (TC) p14 rat (Wistar) brain slices.    -   Stimulation paradigm: 40 Hz (consistent with thalamo-cortical        recurrent stimulation) paired pulse stimulation.    -   Perfusion paradigm: epochs carried out in two phases—for every        drug perfusion (say: control, 0.1 uM T30 etc.), the new        perfusion was applied and left to perfuse for 15 minutes before        starting the recording period (15 minutes also), such that the        drug had time to reach its actual concentration and induce its        concentration-dependent effects once recording. Meaning one        perfusion epoch lasted 30 minutes, with the recording only        taking in account the last 15 minutes of its respective        perfusion epoch.

Detailed Methods

Brain Slice Preparation

Male Wistar rats (14-17 day old; 15 individual animals in total) wereanaesthetised using isoflurane: 10 mL 100% w/w isoflurane was applied tothe cotton bed at the bottom of an anaesthesia chamber (glass box20×15×15 cm) where rats were then placed for ˜45 seconds until onset ofanaesthesia. The hind paw of each anaesthetised rat was pinched to checkfor appropriate depth of anaesthesia. Once anaesthesia was confirmed,rats were quickly decapitated before immersing the brain in oxygenatedice-cold artificial cerebrospinal fluid (‘slicing’ aCSF in mmol: 120NaCl, 5 KCl, 20 NaHCO₃, 2.4 CaCl₂, 2 MgSO₄, 1.2 KH₂PO₄, 10 glucose, 6.7HEPES salt and 3.3 HEPES acid; pH: 7.1) for 7-8 minutes, the time takento cut the brain into slices. Para-saggital sections (400 μm thick) werecut from a block of brain containing both Thalamus (VPN) and primarysomato-sensory cortex (barrel field) using a Vibratome (Leica VT1000S)and transferred to a bubbler pot containing aCSF at room temperature(‘recording’ aCSF in mmol: 124 NaCl, 3.7 KCl, 26 NaHCO₃, 2 CaCl₂, 1.3MgSO₄, 1.3 KH₂PO₄ and 10 glucose; pH: 7.1), which was identical to thatwhich was used during electrophysiological recordings and VSDI. Sliceswere left in oxygenated (95% O₂-5% CO₂) ‘recording’ aCSF to recuperatefor at least 1 hour before VSD staining.

VSD Setup

Slices were placed in a dark, high humidity chamber filled with aCSFbubbling with 95% O₂-5% CO₂. The dye solution (4% 0.2 mM styryl dyepyridinium4-[2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide(Di-4-ANEPPS, Invitrogen, Paisley, UK) (Tominaga et al., 2000) in aCSF46%, fetal bovine serum 46%, DMSO 3.5% and cremophore EL 0.4%) was thenapplied to the slices as previously described (Badin et al., 2013). Whenstarting VSD recordings, slices were placed in the recording bath on asmall piece of filter paper to keep slice alive and was weighed downappropriately using a home-made plastic grid placed atop the slice.Because of the fluorescent VSD, all of the handling of slices during andafter staining with Di-4-ANEPPS was carried out in almost completedarkness in order to keep the detrimental effects of photo-toxicity andbleaching to a minimum. VPN (where stimulating electrodes were placed)was identified with respect to distance from the tip of the hippocampusand to the side of the internal capsule.

Stimulating electrodes, with impedance (measured at 1000 Hz): 500 kΩ,were placed in VPN, where paired-pulse stimulations (2×100 μs induration; 25 milliseconds inter-stimulus interval—ISI—paired-pulse at 40Hz) were triggered to evoke fast-paced propagating waves of activity inthe innervated barrels using Spike 2 V6.0 (CED Ltd, Cambridge, UK) withrespect to appropriate ISI. Such transient ‘neuronal assemblies’ wererecorded by acquiring 16-bit images with a 1 ms resolution using MiCAMUltima ultra-fast imaging system coupled to a digital camera (BrainVision MiCAM Ultima R3-V20 Master) with Ultima 2004/08 imaging software(Brain Vision). Light was generated using an Osram halogen xenophot64634 HLX EFR Display/Optic lamp and was filtered to emit green light(530±10 nm) using a MHF-G150LR (Moritex Corporation). The emittedfluorescence was passed through a dichroic mirror and a >590 nmhigh-pass filter as described previously (Collins et al., 2007;Devonshire et al., 2010a; Devonshire et al., 2010b; Grandy et al., 2012;Mann et al., 2005).

Drug Preparation & Application

T30 and NBP-14 solutions were prepared fresh at the start of eachexperiment, stock solution aliquots were added to ‘recording’ aCSF asappropriate and bath applied at a constant rate of 1.5 mL per minperfusion using a Minipulse 3 pump (Gilson Scientific Ltd, Bedfordshire,UK). Perfusion conditions were split in 2: the first part consisted of a15 minute perfusion with no recording taking place, such that theappropriate concentration could be achieved in the recording bath beforestarting the second part of the perfusion condition—where the recordingtook place for the next 15 minutes of perfusion (30 averagedsnapshots)—giving a total of 30 minutes per perfusion condition.

Data Analysis and Statistics

VSDI produced 4×4 mm (100×100 pixels) 2-Dimensional images from whichcritical data were extracted such as the time-course of activation,spread and intensity of the overall elicited signal. For each VSDIexperiment, each snapshot's data between 0 and 200 ms after stimulation,encapsulating the peak response, had their parameters measured andaveraged for each condition (total of 30 snapshots per condition forboth T30 and T30 v NBP-14 experiments). In order to achieve this, aregion of interest (ROI) was selected over the active area, whichencompassed the width of the maximum response after it had been filteredwith a threshold that isolated active pixels as those showing activitygreater than 20% of the maximum activity recorded within that region ofinterest. Such data were then compiled to produce detailed quantitativegraphs of the extent of activation intensity (FIG. 16, 17, 18) as wellas qualitative ‘space-time’ maps (FIG. 19) to measure the effectsrecorded as well as to produce accurate visual representations of thespatio-temporal data acquired. All statistical tests (Analysis ofVariance—ANOVA) were performed using non-linear mixed effects modelsfitted to the data using R Studio while all the data handling andanalysis was performed using Mathematica 8 (Wolfram Research, USA). Forall statistical tests P<0.05 was considered significant. Data areexpressed as mean±S.E.M.

Results and Discussion

FIG. 16 shows the effect of increasing dose of T30 on (A) intensity ofemitted fluorescence, as well as (B) spread of active pixels. Each pixelhas dimensions 40×40 μm meaning its specific fluorescence arises fromthe activity of less than 10 independent neurons. As the T30concentration increases within the recording bath, the intensity offluorescence given off by active pixels diminishes. This indicates ade-synchronisation of triggered neuronal population activity, assizeable and simultaneous neuronal membrane depolarisation from a singlepixel amounts to a higher fluorescence reading, the opposite effect isseen here (less synchrony). Additionally, here a 40 Hz paired-pulsestimulation paradigm is used, as can be seen from the Intensity graph(FIG. 16A). The results show that not only is the overall fluorescencereduced as a result of T30 treatment, but that the second pulse (whichis triggered at 75 ms, 25 ms after the first one—40 Hz paired pulse)shows a much reduced facilitation compared to the magnitude of theoriginal pulse.

Furthermore, as can be seen from the spread graph (FIG. 16B), the spreadof cortical assemblies is not significantly affected by T30 treatmentuntil very high levels (5 uM) are achieved, this corroborates the theorythat T30 acts as a modulating agent on whole cortical networks. It isalso important to keep in mind that the different parameters highlightedby VSDI, such as the velocity of propagation, the spread and theintensity of fluorescence, often rely on underlying principles ofcortical dynamics which are not necessarily related, meaning each ofthese parameters must be analysed separately and that their results mustat first be interpreted independently from each other.

FIG. 17 shows the effect of increasing T30 application upon the dynamicsof assembly initiation and propagation were investigated in greaterdetail. FIG. 17 shows that T30 induces a reduction in neuronal activitypropagation speed (here acquired as the slope of the rise phase),consistent with a decrease in the synchrony of cortical networks, asshown in FIG. 16, with a maximum of a 3.5-fold decrease in propagationspeed under 5 μM T30 treatment. Resulting from the above, and previousexperiments, it was concluded that the 5 μM T30 concentration was toohigh as it was probably inducing sufficient calcium influx to be at thethreshold for inactivation of the ion channel, thus leading to a mixtureof excitatory/inhibitory effects depending on the sensitivity of theparticular preparation, as reported previously (Bon and Greenfield,2003). The inventors therefore used a lower, yet sufficiently potent,concentration, 1 μM T30, to carry out the subsequent experimentsexploring the possible antagonistic effects of NBP14. This T30concentration, although still quite high, was chosen because of thenature of the present study, where an inevitable dilution effect is tobe expected as the peptide penetrates the brain slice. Meanwhile, forNPB14, they used increasing concentrations of: 0.1, 5, 100 & 300 nM,i.e. 2 to 4 orders of magnitude lower than the concentrations of T30,since previous in vitro studies in PC 12 cells had indicated a farhigher affinity for alpha-7 nicotinic acetylcholine receptors comparedto that of T30.

FIG. 18 shows antagonism of T30 effects by increasing concentrations ofNBP-14. The Figure shows the antagonistic nature of NBP-14 on themodulatory effects induced by T30 perfusion on neuronal populations. Animportant consideration is the dilution factor as T30 penetrates slices,including all physiological processes potentially still present andactive within brain slices, such as proteases, neurotransmitter uptakeand the density of the extracellular matrix; it therefore seems highlyprobable that a concentration of 1 μM T30 would take time to induce itsfull effects (45-60 minutes, as suggested by the data presented above).Bearing this in mind, the effects of T30 become apparent during the 5 nMNBP-14 perfusion (orange line/bar), after which the trend induced by T30is reversed back towards baseline (blue bar/line).

It is important to also note that NBP-14 has been shown to be inert,never inducing any modulatory effects on its own, implying that theeffects seen here are at first attributable to T30, and their reversalto antagonism by increasing concentrations of NBP-14. Importantly, thevast majority of T14 effects are reduced back to control levels underthe 300 nM NBP-14 perfusion, while T30 is perfused at a concentration of1000 nM. This suggests a significantly higher affinity of NBP-14 for itstarget compared to T30.

FIG. 19 shows qualitative results from the three experiments (ie left,centre and right-hand columns) where T30 (1 μM) effects were testedagainst increasing concentrations of NBP-14 (0.1, 5, 100 & 300 nM). Toppanel shows the two main averaged-data graphs: left—Intensity offluorescence signal, and right—spread of evoked assemblies. Bottom panelshows ‘space-time’ maps mapping the activity of a row of pixels lyingover the area of interest (y-axis) over time (310 ms total, x-axis) foreach perfusion conditions. The drastic reduction in fluorescenceintensity as a result of T30 and NBP-14 co-perfusion is clearly evident,as it is on the Intensity graph. Note: the space-time maps are labelledwith their respective perfusion (left), and are colour-coded to theircorresponding traces both in the intensity (right) and spread (left)graphs.

REFERENCES

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Example 16 Effects of NBP 14 in the Freely Moving Rat

Background

Unlike animal models for Alzheimer's disease, the rat model forhemi-Parkinsonism is very well established and readily quantifiable.Accordingly, a unilateral intra striatum injection of the T30 wasadministered to observe any behavioural effects of the toxin. In asubsequent experiment, the potential protective effects of NBP14 wereobserved against the well-known neurotoxin 6-hydroxydopamine (6-OHDA),which led to DA neuron loss on the injected side whilst sparing thecontralateral DA neurons. NBP-14 was administered via implanted cannulainto the medial forebrain bundle (MFB). 6-hydroxydopamine was injectedat 10 mg/kg.

Detailed Methods

Animals are anaesthetized using Ketamine (10%; 0.1 ml/kg body weight)and Xylazine (2%; 0.01 ml/kg). The animals are then stereotacticallyinjected into the MFB with 2 μL 6-OHDA at a concentration of 20 mg/ml in0.02% ascorbic acid. Lesion coordinates are set according to bregma anddura in cm: L—1.7 mm; AP—3.6 mm; DV—8.0 mm. Following the injection(injection rate 2 μl/5 min), the injecting needle is left for another 1minute to avoid back flow and then slowly retracted.

Paw Placement Test (Cylinder Test): This test assesses a rat'sindependent forelimb use to support the body against the walls of acylindrical enclosure. The test takes advantage of the animals' innatedrive to explore a novel environment by standing on the hindlimbs andleaning towards the enclosing walls. To perform this test, rats areplaced individually in a glass cylinder (21 cm diameter, 34 cm height)and wall exploration is recorded for 3 minutes. No habituation to thecylinder prior to recording is allowed. Wall exploration is expressed interms of the ratio between the intact (R) and impaired legs (L) andcalculated as the values of intact right+both forelimbs divided into thevalues of impaired left+both forelimbs (R/L). The paw placement test isconducted on Day −1 to obtain baseline data, on Day 1 for selection andon days 2.

Selection criteria (Day1): According to Paw placement test all animalswith statistically significant difference between paws will be includedin the study (ratio between the intact (R) and impaired legs (L) isexpressed as the values of intact right+both forelimbs divided into thevalues of impaired left+both forelimbs).

The results from all tests will be presented as MEAN group value±SEM.Analysis of the data by one-way ANOVA following by Tukey test will beapplied to determine significance of treatment effects. This study wasperformed following approval of an application form submitted to theCommittee for Ethical Conduct in the Care and Use of Laboratory Animalsthat states that the present study complied with the rules andregulations set forth.

FIG. 20 summarises the procedure followed during the in vivo testing ofNBP-14.

Results

Effects NBP14

Analysis of the paw placement R/L ratio reflects unilateral injury ofmotor function. On day 2, i.e. two days post 6-OHDA injection and oneday after injection of NBP-14, there was a significant difference in theR/L ratio of paw placement between 6-OHDA vehicle and treated: 7.54±1.63vs. 3.62±0.55, respectively (p<0.05). Treatment with NBP-14 improvedmobility of impaired forelimb after one dosing as was shown in the pawplacement test (FIG. 21).

Example 17 Effects of T30 and NBP14 on APP and Amyloid

Background

It is has already been established that an excess of calcium can triggerabnormal cleavage of Amyloid Precursor Protein (APP) and hence Amyloidbeta (Aβ) release (Hartigan & Johnson, 1999; Cai et al., 2012). Sincethe inventors have shown that T30 increases calcium influx by about 70%in PC12 cells, it is possible that such a calcium increase will triggerthe production of amyloid and a consequent decrease in the full lengthAPP molecule.

Detailed Methodology: Detection of APP

Protocol for Solubilizing Protein

PC12 cells are plated with growth medium in Petri Dishes for a week inorder to have enough protein to detect APP in PC12 membranes and treatedfor 1 hour with T30 and NBP-14 before solubilizing the protein. Once thecells have grown until 90% confluence, the growth medium is removed andcells are re-suspended in 2 ml of HBSS. The cells suspension istransferred to a 15 ml tube and centrifuge 5 minutes at 1000 rpm. Thenthe supernatant is discarded and the pellet is re-suspended in Lysisbuffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA; pH 8) plusprotease inhibitors (1 μl:1 ml PMSF and 3 μl:1 ml Aprotinin) andtriturated by using a Polytron for 10 seconds. Subsequently, thetriturated pellet is distributed in 1.5 ml eppendorfs and rotated orshaken for 2 h at 4° C. After 2 h, the eppendorfs are centrifuged at15000 rpm for 20 minutes and the supernatant is kept. The Bradfordreagent is used to quantify the protein contained in each eppendorf.

Protocol for Electrophoresis

For APP detection, an aliquot of 25 μg of protein is used. Beforestarting the protocol the reagents are prepared as follows:

Lower Gel (w %) (20 Min to Polymerize)

For 10 ml (2 gels):3.6 ml H2O MQ, 2.42 ml Acrilamide and 1.3 mlBis-Acrilamide, 2.5 ml Tris-HCl1.5 M pH 8.8, 0.11 ml SDS 10%, 0.06 mlAmmonium persulfate 10%, 6.67 μl TEMED (last ingredient).

Upper Gel (5%) (20 Min to Polymerize)

For 5 ml (2 gels): 3.67 ml H2O MQ, 0.48 ml Acrilamide and 0.26 mlBis-Acrilamide, 0.625 ml Tris-hCl 1 M pH 6.8, 0.05 ml SDS 10%, 25 μlAmonium persulfate 10%, 5 μl TEMED.

Tris-HCl 1.5 M pH 8.8

For 100 ml: 18.16 gr Tris Base, qsp 100 ml H2O MQ, pH 8.8.

Tris-HCl 1 M pH 6.8

For 100 ml: 12.1 gr Tris Base, qsp 100 ml H2O MQ, pH 6.8.

Sample Buffer (4×)

For 8 ml: 3.2 ml SDS 10%, 1.6 ml Glicerol, 2 ml Tris-HCl 1 M pH 6.8, 0.8ml B-Mercaptoethanol, 0.4 ml Bromophenol Blue 0.1% or Red. (Use 1× forexperiment)

Running Buffer (10×)

For 1 L: 30.3 g Tris base, 144 gr Glycine, 10 gr SDS, qsp 1 L H2O MQ.(Use 1× for experiment)

The steps for electrophoresis are the following:

-   a) Prepare the lower and the upper acrylamide gels. The % for APP    gel is 10% lower gel and 5% stacking gel.-   b) Prepare 24 μl of sample at a concentration of 25 μg (determined    by the Bradford Assay) (6 μl SB 4× containing    β-mercaptoethanol+protein+lisis) and boil them at 100° C. for 5    minutes to denaturalize them.-   c) Put the protein marker and the samples in the wells of the gel    (20-30 μL).-   d) Proceed to Migration: 35 mA (nearly 1 hour).

Protocol for Western Blot

Before starting the protocol the reagents are prepared as follows:

Transfer buffer (1×)

For 1 L: 3.03 g Tris base, 14.4 gr Glycine, 200 ml Methanol, qsp 1 L H2OMQ.

TBS buffer (4×)

For 1 L: 24.25 gr Tris base, 60 gr NaCl, qsp 1 L H2O MQ, pH 7.5.

TBS-Tween Buffer

For 1 L: 250 ml TBS 4×, 0.5 ml Tween 20, qsp 1 L H2O MQ.

There are 2 steps to follow, the electrotransfer and the immunodetectionof protein, see the steps below:

1) Electro Transfer

-   a) Activate the PVDF membrane: 1 minute in MeOH and 2 minutes in MQ    H2O.-   b) Put the PVDF membranes, papers and sponges in Transfer buffer    during 10 minutes.-   c) Prepare the sandwich and proceed with the transfer of the    proteins from the gel to the PDVF membrane: 0.2 A during 2 hours.

2) Immunodetection of Proteins

-   a) Block the inespecific sites of the membrane with milk 5%    (dissolve it in TBS-T).-   b) Incubation with the primary antibody (dissolved in TBST/milk 5%):    Anti-Amyloid Precursor Protein (ab2072, rabbit) at a dilution 1:500    (20 μl in 10 ml), over-night at 4° C.-   c) Wash the membrane with TBS-T (5 min x2).-   d) Incubation with the secondary antibody dissolved in TBS-T:    Anti-rabbit-HRP (goat) dilution 1:5000 (20 μl in 10 ml) for 45    minutes at room temperature.-   e) Wash the membrane with TBS-T (5 min x2+10 min x1).-   f) Take a picture with Chemibox option (white light) to see the    position of marker bands.-   g) Add ECL reagent (HRP) for antibody detection (iml of each    component) and take several pictures with Chemibox option (no    light).

Detailed Methodology: Detection of A1342 in Cell Culture Media

After 1 hour of treatment, the culture media was collected and dilutedto 1:100, using culture media as diluent. Four repeats of each dilutedsample were then placed in the plate provided by the ELISA detectionkit, from AnaSpec (Fremont, Calif., USA). The detection was then carriedout following the manufacturer's protocol. Briefly, the sample wasincubated for 4 h in presence of 50 μl of detection antibody. The platewas then washed seven times with the washing solution, the samples werethen incubated with the 3,3′,5,5′-Tetramethylbenzidine (TMB) providedwith the kit for 15 min. after the sample revelation the reaction wasstopped with the stop solution and the optical density was read at 450nm.

FIG. 22 is a diagram showing the cascade of events resulting from theeffect of T30 in a cell: (1) T30 binds to the allosteric site of thereceptor to enhance the opening of the channel for Ca²⁺ influx into thecell (Greenfield et al. 2004). (2) Calcium entry induces depolarizationand opening of the voltage-dependent (L-VOCC) channel allowing stillmore Ca²⁺ into the cell (Dickinson et al., 2007). (3) This raisedintracellular calcium induces an increase in AChE G₄ release thatincludes T30 (Greenfield, 2013). (4) Calcium also induces upregulationof the α7 nicotinic receptor that will allow more Ca²⁺ get in the cellby providing still more targets for T30 (Bond et al.,2009). (5) Calciumactivates enzymes (ie GSK-3) that will (a) increase Tau, (b) activateγ-secretase/β-secretase that will trigger cleavage of extracellulartoxic Amyloid that (c) together with T30 will promote a still furthertoxic amount of Ca²⁺ into the cell. (Hartigan & Johnson (1999), Cai etal. (2012), Garcia-Ratés et al (2013)).

Accordingly, using immunodetection, the inventors have determined (i)APP levels and (ii) release of amyloid following administration of T30(5 uM) and NBP14 (0.5 μM).

Results

-   -   (i) As shown in FIG. 23, T30 reduces levels of full length APP        in PC12 cell membranes, an effect reversed by NBP14. The values        are: Control (100%±10.98); T30 5 μM (67.82%±6.23%) and        T30+NBP-14 0.5 μM (126%±1.12%).

FIG. 24 shows immunodetection by western blot represented in the graph.The 3 different treatments show different levels of expression of APP(values represented in FIG. 24). For each condition protein is correctedby levels of GAPDH.

The production of APP is reduced by T30 peptide, an effect which isreversed by NBP-14. This suggests that APP could be cleaved, releasingAmyloid-β1-42 peptide (Aβ42).

-   -   (ii) In order to determine the release of Aβ42 we used an ELISA        kit, commercially available, measuring Aβ42 present in solution.        This test showed that T30 increases the release of Aβ42 up to        approximately 175% compared to control and NBP-14 brings the        release of Aβ42 to a value close to control (see FIG. 25).

1-3. (canceled)
 4. A cyclic polypeptide, derivative or analogue thereof,comprising an amino acid sequence derived from the C-terminus ofacetylcholinesterase (AChE), or a truncation thereof. 5-8. (canceled) 9.The cyclic polypeptide, derivative or analogue thereof according toclaim 4, wherein the cyclic polypeptide, derivative or analogue thereofcomprises an amino acid sequence derived from the C-terminus of tailedacetylcholinesterase (T-AChE), or a truncation thereof.
 10. The cyclicpolypeptide, derivative or analogue thereof according to claim 4,wherein the cyclic polypeptide, derivative or analogue thereof comprisesan amino acid sequence derived from the last 300, 200, 100, 50, or 40amino acids forming the C-terminus of acetylcholinesterase, or atruncation thereof, and wherein the acetylcholinesterase comprises anamino acid sequence substantially as set out in SEQ ID No:1. 11-12.(canceled)
 13. The cyclic polypeptide, derivative or analogue thereofaccording to claim 4, wherein the cyclic polypeptide, derivative oranalogue thereof comprises between 8 and 40 amino acid residues, orbetween 10 and 30 amino acids, or between 12 and 20 amino acids.
 14. Thecyclic polypeptide, derivative or analogue thereof according to claim 4,wherein the cyclic polypeptide, derivative or analogue thereof comprisescyclic SEQ ID No:3, 4, 5 or 6, or a functional variant or fragmentthereof.
 15. The cyclic polypeptide, derivative or analogue thereofaccording to claim 4, wherein the cyclic polypeptide, derivative oranalogue thereof comprises cyclic SEQ ID No:6, or a functional variantor fragment thereof, wherein x₁ is a basic amino acid residue,preferably histidine (H); x₂ is a basic amino acid residue, preferablyarginine (R); x₃ is an aromatic amino acid residue, preferablytryptophan (W); x₄ is an amino acid residue having an aliphatic hydroxylside chain, preferably serine (S); and x₅ is tryptophan (W) ormethionine (M).
 16. The cyclic polypeptide, derivative or analoguethereof according to claim 4, wherein the cyclic polypeptide, derivativeor analogue thereof comprises cyclic SEQ ID No:4, or a functionalvariant or fragment thereof.
 17. The cyclic polypeptide, derivative, oranalogue thereof according to claim 4, wherein the cyclic polypeptide,derivative, or analogue thereof is a selective allosteric modulator ofthe α7 nicotinic-receptor, which prevents the additional influx ofcalcium through an allosteric site of the α7 nicotinic-receptor, andoutcompetes binding for β-amyloid. 18-26. (canceled)
 27. Apharmaceutical composition comprising a therapeutically effective amountof the cyclic polypeptide, derivative or analogue thereof according toclaim 4, and a pharmaceutically acceptable vehicle.
 28. (canceled) 29.The pharmaceutical composition according to claim 27 wherein thepolypeptide, derivative or analogue thereof comprises cyclic SEQ IDNo:4, or a functional variant or fragment thereof.
 30. A method of usingthe cyclic polypeptide, derivative or analogue thereof according toclaim 4, in an in vitro or ex vivo analytical method for investigatingthe allosteric site of the α7 nicotinic-receptor, comprising the step ofpreventing additional influx of calcium through the α7 nicotinicreceptor.
 31. (canceled)
 32. A method of treating, ameliorating orpreventing a neurodegenerative disorder in a subject, the methodcomprising, administering to a subject in need of such treatment, atherapeutically effective amount of the cyclic polypeptide, derivativeor analogue thereof according to claim
 4. 33. The method according toclaim 32, wherein the polypeptide, derivative or analogue thereofcomprises cyclic SEQ ID No:4, or a functional variant or fragmentthereof.
 34. The method according to claim 32, wherein theneurodegenerative disorder is selected from a group consisting ofAlzheimer's disease; Parkinson's disease; Huntington's disease; MotorNeurone disease; Spinocerebellar type 1, type 2, and type 3; AmyotrophicLateral Sclerosis (ALS); Frontotemporal Dementia; and Schizophrenia. 35.A receptor allosteric modulator comprising a cyclic polypeptide,derivative or analogue thereof.
 36. A process for making thepharmaceutical composition according to claim 27, comprising the step ofcontacting the cyclic polypeptide, derivative, or analogue thereof witha pharmaceutically acceptable vehicle.